Adenoviral vectors for treatment of hemophilia

ABSTRACT

An adenoviral vector including at least one DNA sequence encoding a clotting factor, such as, for example, Factor VIII, or Factor IX. Such vectors may be administered to a host in an amount effective to treat hemophilia in the host. The vectors infect hepatocytes very efficiently, whereby the hepatocytes express the DNA sequence encoding the clotting factor.

This application is a continuation-in-part of application Ser. No.08/218,335, filed Mar. 25, 1994, now abandoned, which is acontinuation-in-part of application Ser. No. 074,920, filed Jun. 10,1993, now abandoned.

FIELD OF THE INVENTION

This invention relates to adenoviral vectors. More particularly, thisinvention relates to adenoviral vectors which may be employed in thetreatment of hemophilia.

BACKGROUND OF THE INVENTION

Hemophilias A and B are X-linked, recessive bleeding disorders caused bydeficiencies of clotting Factors VIII and IX, respectively. In theUnited States there are approximately 17,000 patients with hemophilia Aand 2,800 with hemophilia B. The clinical presentations for bothhemophilias are characterized by episodes of spontaneous and prolongedbleeding. Patients frequently suffer joint bleeds which lead to adisabling arthropathy. Current treatment is directed at stopping thebleeding episodes with intravenous infusions of plasma-derived clottingfactors or, for hemophilia A, recombinant Factor VIII. However, therapyis limited by the availability of clotting factors, their shorthalf-lives in vivo, and the high cost of treatment, which can approach100,000 dollars per year.

Gene therapy offers the promise of a new method of treating hemophilia.Several groups of researchers have conducted research with retroviralvectors containing RNA encoding Factor VIII and Factor IX. Prior toApplicants' invention, virtually every attempt to produce therapeuticlevels of these factors in vivo with such vectors, however, has beenunsuccessful. The cDNA and the RNA for Factor VIII has been particularlydifficult to work with.

Hoeben et al., J. Biol. Chem, Vol. 265, pgs 7318-7323 (1990) and Israelet al. Blood, Vol. 75, No. 5, pgs. 1074-1080 (Mar. 1, 1990) describe theinfection of mouse fibroblasts in vitro with retroviral vectorsincluding DNA (RNA) encoding B-domain deleted human Factor VIII.Although such infected cells were found to express functional humanFactor VIII in vitro, the protein was expressed at low levels.

Recently, Hoeben et al., Human Gene Therapy, Vol. 4, pgs 179-186 (1993)infected fibroblasts with retroviral vectors including DNA encodinghuman Factor VIII. These cells then were implanted into immune-deficientmice. Although cells recovered from the implants up to 2 monthspost-implantation still had the capacity to secrete Factor VIII whenregrown in tissue culture, human Factor VIII was not detected in plasmasamples of the recipient mice.

Lynch et al., Human Gene Therapy, Vol. 4, pgs. 259-272 (1993), describesthe transfection of PE501 packaging cells with the plasmid forms ofretroviral vectors including human Factor VIII cDNA. The virus washarvested, and used to infect PA317 amphotropic retrovirus packagingcells. The infected cells, however, produced human Factor VIII and virustiter in an amount which was about two orders of magnitude lower thanthose from similar retroviral vectors containing other cDNAs. Lynch etal. also observed a 100-fold lower accumulation of vector RNAscontaining the human Factor VIII sequences in comparison to vectorscontaining other cDNA sequences.

Lynch et al. also reported the following difficulties in working withFactor VIII. High titer human Factor VIII-containing retroviral vectorstocks are difficult to generate, and retroviral vectors containingFactor VIII cDNA sequences tend to rearrange and/or delete portions ofthe Factor VIII cDNA sequences. In addition, Factor VIII mRNA isinherently unstable. Also, the B-domain deleted Factor-VIII codingregion contains a 1.2 kb RNA accumulation inhibitory signal.

Thus, there have been significant problems in working with retroviralapproaches to gene therapy with Factor VIII and that only limitedexpression has been achieved prior to Applicants' invention.

Researchers also have experienced significant difficulties in attemptingto achieve therapeutic levels of Factor IX expression with retroviralvectors prior to Applicants' invention.

Palmer et al., Blood, Vol. 73, No. 2, pgs. 438-445 (February 1989)discloses the transduction of human skin fibroblasts with retroviralvectors including DNA (RNA) encoding human Factor IX. Such transformedfibroblasts then were given to rats and to nude mice. Although suchfibroblasts were found to transiently express human Factor IX in theanimal blood in amounts up to 190 ng/ml, this amount is not generallyconsidered to be at a therapeutic level.

Scharfmann et al., Proc. Nat. Acad. Sci., Vol. 88, pgs. 4626-4630 (June1991) discloses the transduction of mouse fibroblast implants with aretroviral vector including a B-galactosidase gene under the control ofthe dihydrofolate reductase (DHFR) promoter. Such fibroblasts then weregrafted into mice, and expression of the β-galactosidase gene for up tosixty days was obtained. Scharfmann et al. also disclose fibroblaststransduced with canine Factor IX, but they only obtained short-term andnon-therapeutic levels of expression.

Dai et al., Proc. Nat. Acad. Sci., Vol. 89, pgs. 10892-10895 (November1992) discloses the transfection of mouse primary myoblasts withretroviral vectors including canine Factor IX DNA under the control of amouse muscle creatine kinase enhancer and a human cytomegaloviruspromoter. The transfected myoblasts then were injected into the hindlegs of mice. Expression of canine Factor IX over a period of 6 monthswas obtained; however, the steady-state levels of Factor IX secretedinto the plasma (10 ng/ml for 10⁷ injected cells) are not sufficient tobe of therapeutic value.

Gerrard et al., Nature Genetics, Vol. 3, pgs. 180-183 (February 1993),discloses the transfection of primary human keratinocytes with aretroviral vector including a human Factor IX gene under the control ofthe retroviral LTR. The transformed keratinocytes then were transplantedinto nude mice, and human Factor IX was detected in the bloodstream forabout 1 week. The amounts of Factor IX, however, were about 2.5 ng/ml,or about 1% of a therapeutic dose.

Kay et al., Science, Vol. 262, pgs. 117-119 (Oct. 1, 1993) discloses thedirect infusion of retroviral vectors including Factor IX DNA into theportal vasculature of dogs following partial hepatectomy. The animalsexpressed low levels of canine Factor IX for more than 5 months.Although such expression of Factor IX resulted in reductions of wholeblood clotting and partial thromboplastin times of the treated animals,the authors stated that increased levels of Factor IX must first beachieved before the technique could be applied to humans.

Zhou et al., Science in China, Vol. 36, No. 9, pgs. 33-41 (September1993) discloses the transfection of rabbit skin fibroblasts withretroviral vectors including DNA encoding human Factor IX. Thefibroblasts then were implanted into rabbits as autografts orallografts. Expression of the human Factor IX was maintained in therabbits for over 10 months. Factor IX levels in the rabbit plasma of upto 480 ng/ml were claimed to have been achieved; however, the assay usedto measure Factor IX employed an anti-rabbit antibody that had thepotential of generating false positive results.

Lu et al., Science in China, Vol. 36, No. 11, pgs. 1341-1351 (November1993) and Hsueh et al., Human Gene Therapy, Vol. 3, pgs. 543-552 (1992)discloses a human gene therapy trial in which human skin fibroblastswere taken from two hemophiliac patients, and transfected withretroviral vectors including DNA encoding human Factor IX. The cellsthen were pooled and embedded in a collagen mixture, and the cells thenwere injected into the patients. In one patient, the concentration ofhuman Factor IX increased from 71 ng/ml to 220 ng/ml, with a maximumlevel of 245 ng/ml. The clotting activity of this patient increased from2.9% to 6.3% of normal. In the other patient, the plasma level of FactorIX increased from 130 ng/ml to 250 ng/ml, and was maintained at a levelof 220 ng/ml for 51/2 months; however, the clotting activity has notincreased. Lack of pretreatment Factor IX data on these patients makesit difficult to interpret the small increases in Factor IX seen intreatment.

The conclusion to be drawn from scientific literature at the time ofApplicants' invention, on the attempts to use retroviruses in genetherapy for hemophilia A and hemophilia B is that, in spite of a veryconcerted effort and numerous attempts, by and large the field hasfailed to produce retroviral vectors that can be used to achievetherapeutic levels of expression of human Factor VIII or human Factor IXin vivo. Working with Factor VIII has been especially difficult, and theresults have been unsatisfactory. The experimental strategies describedabove are laborious and clinically invasive.

Adenoviral vectors offer another approach to gene therapy. Adenovirusgenomes are linear, double-stranded DNA molecules of approximately 36kilobase pairs. Each extremity of the viral genome has a short sequenceknown as the inverted terminal repeat (or ITR), which is necessary forviral replication. The well-characterized molecular genetics ofadenovirus render it an advantageous vector for gene transfer. Portionsof the viral genome can be substituted with DNA of foreign origin. Inaddition, recombinant adenoviruses are structurally stable and norearranged viruses are observed after extensive amplification.

Recombinant adenoviruses have been used as efficient vectors for genetransfer into a number of cell types. There are several reports ofhepatocyte transduction: Jaffe et al., Nature Genetics, Vol. 1, pgs.372-378 (1992) (alpha-1-antitrypsin); Li et al., Human Gene Therapy,Vol. 4, pgs. 403-409 (1993) (beta-galactosidase); Stratford-Perricaudetet al., Human Gene Therapy, Vol. 1, pgs. 241-256 (1990) (ornithinetranscarbamylase); Smith, et al., Nature Genetics, Vol. 5, pgs. 397-402(1993) (Factor IX); and J. Am. Med. Assoc., Vol. 269, No. 7, pg. 838(Feb. 17, 1993) (marker protein).

Because Factor VIII is synthesized largely in hepatocytes (Kelly et al.Br. J. Haemat., Vol. 56, pgs. 535-543 (1984); Wion et al., Nature, Vol.317, pgs. 726-729 (1985); Zelechovska et al. Nature, Vol. 317, pgs.729-732 (1985)), transduction of hepatocytes with a FactorVIII-containing recombinant adenovirus, resulting in the expression ofFactor VIII protein in vivo, may be an effective gene therapy-basedtreatment for hemophilia A.

The inventors have discovered how to produce high titer, stable,adenoviral vectors that produce therapeutic levels of clotting factorswhen administered to an animal host. These vectors mediate gene transferin vivo and will enable treatment protocols to be much less laboriousand invasive than those previously described.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described with respect to the drawings,wherein:

FIG. 1 is a map of plasmid pG1;

FIG. 2 is a map of plasmid pG1H9;

FIG. 3 is a map of plasmid phfacIX;

FIG. 4 is a map of plasmid pG1H9B;

FIG. 5 is a map of pAvS6;

FIG. 6 is the human Factor IX cDNA sequence;

FIG. 7 is a map of pAvS6H9B;

FIG. 8 is a schematic of the generation of Av1H9B;

FIG. 9 is a graph of plasma levels of Factor IX in mice givenintraparenchymal or portal vein injections of Av1H9B;

FIG. 10 is an autoradiogram of a Southern analysis to determine thepresence of Factor IX DNA in mouse liver;

FIG. 11 is a map of plasmid pMT2LA;

FIG. 12 is the sequence of B-domain deleted human Factor VIII cDNA;

FIG. 13 is a map of plasmid pAvS6H81;

FIG. 14 is a schematic of the construction of Av1H81;

FIG. 15 is a schematic of the construction of plasmid pAvALH81;

FIG. 16 is a map of plasmid pAvALH81;

FIG. 17 is a schematic of the construction of plasmid pAvAPH81;

FIG. 18 is a map of plasmid pAvAPH81;

FIG. 19 is a schematic of the construction of plasmid pAvALPH81;

FIG. 20 is a map of plasmid pAvALAPH81;

FIG. 21 is a schematic of the generation of Av1ALH81;

FIG. 22 is a photograph of an ethidium bromide stained gel showingrestriction digestion analysis of Av1ALH81 DNA;

FIG. 23 is a schematic of adenoviral vectors Ads-dl327, Av1ALH81, andAv1ALAPH81;

FIG. 24 is a standard log-log curve of a human Factor VIII-specificELISA assay;

FIGS. 25 and 26 are graphs of the amounts of human Factor VIII in mouseplasma over time in two separate experiments;

FIG. 27 is a graph of in vivo expression of human Factor VIII in mouseplasma over time after injection of various doses of Av1ALH81;

FIGS. 28 and 29 are graphs of human Factor VIII half-life inexperimental mice and control mice;

FIG. 30 is a graph of in vivo expression of human Factor VIII in micegiven 4×10⁹ pfu of Av1ALH81;

FIG. 31 is a map of plasmid pBLSKH9CI;

FIG. 32 is a map of plasmid pBLSKH9D;

FIG. 33 is a map of Plasmid pBLH9CINT;

FIG. 34 is a map of plasmid pBLH9EINT;

FIG. 35 is a map of plasmid pBLH9E;

FIG. 36 is a map of plasmid pBLH9F:

FIG. 37 is a map of plasmid pAV1H9D;

FIG. 38 is a map of plasmid pAV1H9ER;

FIG. 39 is a map of plasmid pAV1H9FR;

FIG. 40 is a graph of Factor IX expression in mice treated with Av1H9B,Av1H9D, Av1H9ER and Av1H9FR;

FIG. 41 is a graph of Factor IX expression in mice treated with 1×10⁹pfu of Av1H9B, Av1H9D, or Av1H9ER;

FIG. 42 is a graph of Factor IX expression in mice treated with 1×10⁹pfu of Av1H9FR;

FIG. 43 is a graph of in vitro expression of Factor IX in HepG2 and HeLacells, and of in vivo expression of Factor IX in mice treated with 2×10⁸pfu of Av1H9B, Av1H9D, or Av1H9ER. In each group of 3 bars, the leftmostbar represents data for Av1H9B, the middle bar, Av1H9D, and therightmost bar, Av1H9ER.

FIG. 44 is an immunoprecipitation purification of B-domain deletedFactor VIII produced in HepG2 cells transduced with Av1ALAPH81,Av1ALH81, or Av1ALH9B, or mock infected cells;

FIG. 45 is a graph of the expression of human Factor VIII in the plasmaof mice which received 4×10⁹ pfu of Av1ALAPH81, Av1ALH81, or Av1ALH9B;

FIG. 46A is a Southern blot analysis of DNA from the livers of micewhich received Av1ALH81 or Av1ALAPH81;

FIG. 46B is an autoradiograph of an RNase protection analysis of RNAfrom the livers of mice which received Av1ALAPH81 or Av1ALH81;

FIG. 46C is a schematic of a Factor VIII probe template and thecomplementary Factor VIII mRNA fragment;

FIG. 47A is an autoradiograph of RNase protection analysis of RNA fromthe livers of mice which received Av1ALH81 or Av1ALAPH81, to determinetranscription initiation and splicing efficiency of vector-derived RNA;

FIG. 47B is a schematic depicting the probes employed in FIG. 47A;

FIG. 48A is a Southern blot analysis of DNA isolated from the livers,lungs, and spleens of mice that received Av1ALAPH81;

FIG. 48B is an autoradiograph of RNase protection analysis of RNAisolated from the livers, lungs, and spleens of mice that receivedAv1ALAPH81;

FIG. 48C is a schematic of the probe template and complementary FactorVIII mRNA fragment employed in FIG. 48B;

FIG. 49 is a graph of human Factor VIII expression in mice afteradministration of 5×10⁸ pfu of Av1ALAPH81 OR Av1ALH81;

FIGS. 50A and 50B are graphs of dose response curves for the time courseof human Factor VIII expression in mice that received 4×10⁹ pfu; 1×10⁹pfu; 5×10⁸ pfu; 2×10⁸ pfu; or 5×10⁷ pfu of Av1ALAPH81;

FIG. 51A is a Southern blot analysis of DNA isolated from the livers ofmice which received 4×10⁹ pfu of Av1ALAPH81;

FIG. 51B is an autoradiograph of RNase protection analysis of RNAisolated from the livers of mice which received 4×10⁹ pfu of Av1ALAPH81;

FIG. 51C is a Southern blot analysis of DNA of the livers of mice whichreceived 5×10⁸ pfu of Av1ALAPH81;

FIG. 51D is an autoradiograph of RNase protection analysis of RNAisolated from the livers of mice which received 5×10⁸ pfu of Av1ALAPH81;

FIG. 52 is a graph showing the half-life of human Factor VIII in plasmasamples of mice which received 1,000 ng of purified full-length humanFactor VIII four months after the mice received 5×10⁸ pfu of Av1ALAPH81,compared with control mice which received no vector;

FIGS. 53A, 53B, 53C and 53D are graphs of the amounts of aspartateaminotransferase, alanine aminotransferase, sorbitol dehydrogenase, andalkaline phosphatase, respectively, which are present in plasma samplesof mice which received 4×10⁹ pfu or 5×10⁸ pfu of Av1ALAPH81, or wereuninjected;

FIG. 54 is a graph of the amount of human Factor VIII present in theplasma of a hemophiliac dog treated with 10¹² pfu of Av1ALAPH81,measured by the Coatest functional assay;

FIG. 55 is a graph of Factor VIII biological activity and of humanFactor VIII antigen levels measured by ELISA in the plasma of thehemophiliac dog treated with 10¹² pfu of Av1ALAPH81;

FIG. 56 shows Southern blot and RNase protection analysis of DNA and RNAisolated from the liver and spleen of the hemophiliac dog treated with10¹² pfu of Av1ALAPH81;

FIG. 57 is a schematic of Ad5-dl327, Av1APH81, Av1AP3'H81, Av1APH8H9,Av1ALAP3'H81, and Av1ALAPH8H9;

FIG. 58 is a graph of expression of human Factor VIII in HepG2 cellstransduced with Av1ALAPH81, Av1ALAP3'H81, Av1APH81, Av1ALH9B,Av1AP3'H81, or Av1APH8H9;

FIG. 59 is a graph of human Factor VIII expression in mice which weregiven 4×10⁹ pfu of Av1ALAP3'H81, Av1APH81, Av1AP3'H81, Av1APH8H9, orAv1ALH9B, or were given 5×10⁸ pfu of Av1ALAPH81;

FIG. 60 is a schematic of adenoviral vectors Av1H9FR, Av1H9F1, andAv1H9F2; and

FIG. 61 is a Southern blot analysis of DNA from the livers of mice whichwere given 5×10⁷ pfu or 1×10⁸ pfu of Av1H9FR, Av1H9F1, or Av1H9F2, withaverage values of plasma human Factor IX from each cohort of mice.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with an aspect of the present invention, there is providedan adenoviral vector including at least one DNA sequence encoding aclotting factor.

The term "DNA sequence encoding a clotting factor" as used herein meansDNA which encodes a full-length clotting factor or a fragment,derivative, or analogue of a clotting factor, i.e., such DNA may be afull-length gene encoding a full-length clotting factor, or a truncatedgene, or a mutated gene encoding a fragment or derivative or analogue ofsuch clotting factor which has clotting factor activity. The term "DNAsequence" refers generally to a polydeoxyribonucleotide molecule andmore specifically to a linear series of deoxyribonucleotides connectedone to the other by phosphodiester bonds between the 3' and 5' carbonsof the adjacent pentoses.

In one embodiment, the DNA sequence encodes Factor VIII or a fragment,derivative, or analogue thereof having Factor VIII clotting activity. Inanother embodiment, the DNA sequence encodes Factor IX or a fragment,derivative, or analogue thereof having Factor IX clotting activity.

The DNA sequence encoding human Factor IX is shown and described in U.S.Pat. No. 4,994,371 issued Feb. 19, 1991 to Davie et al. and EuropeanPatent No. EP 0 107 278 B1 (publication of grant Nov. 15, 1989) toNational Research Development Corporation. DNA sequences encoding FactorVIII and fragments or derivatives thereof are shown and described inU.S. Pat. Nos. 4,757,006 issued Jul. 12, 1988 to Toole, Jr. et al.;4,868,112 issued Sep. 19, 1989 to Toole, Jr.; 5,045,455 issued Sep. 3,1991 to Kuo et al; 5,004,804 issued Apr. 2, 1991 to Kuo et al.;5,112,950 issued May 12, 1992 to Meulien et al.; and 5,149,637 issuedSep. 22, 1992 to Scandella et al.

The inventors have found that, by infecting host cells in vivo withadenoviral vectors including at least one DNA sequence encoding aclotting factor, one is able to achieve expression, in vivo, of theclotting factor, or fragment or derivative or analogue of such clottingfactor having clotting factor activity, at effective therapeutic levels.In general, such effective therapeutic levels are about 5% or greater ofthe normal level of the clotting factor (N. Engl. J. Med., Vol. 328, No.7, pgs. 453-459 (Feb. 18, 1993); Blood, Vol. 74, No. 1, pgs. 207-212(July 1989)). Such levels are, in general, for Factor VIII, about 10ng/ml or greater, and for Factor IX are about 250 ng/ml or greater.

The DNA sequence encoding a clotting factor is under the control of asuitable promoter. Suitable promoters which may be employed include, butare not limited to, adenoviral promoters, such as the adenoviral majorlate promoter; or heterologous promoters, such as the cytomegalovirus(CMV) promoter; the respiratory syncytial virus promoter; the RousSarcoma Virus (RSV) promoter; the albumin promoter; inducible promoters,such as the Mouse Mammary Tumor Virus (MMTV) promoter; themetallothionein promoter; heat shock promoters; the α-1-antitrypsinpromoter; the hepatitis B surface antigen promoter; the transferrinpromoter; the apolipoprotein A-1 promoter; the Factor VIII promoter; andthe Factor IX promoter. It is to be understood, however, that the scopeof the present invention is not to be limited to specific promoters.

In one embodiment, when the DNA sequence encodes Factor VIII or afragment, derivative, or analogue thereof, the promoter controlling theDNA sequence is preferably a tissue-specific promoter, such as, forexample, the mouse albumin promoter, which is active in liver cells.Although the scope of this embodiment is not intended to be limited toany theoretical reasoning, the inventors believe that, by employing atissue-specific promoter, possible Factor VIII toxicity to the producercells is avoided.

When one employs a mouse albumin promoter, which is active in livercells, the adenoviral vectors are preferably grown in cells other thanliver cells. When the generated adenoviral vectors are to beadministered to a host, such vectors are administered to a host by meansknown to those skilled in the art, whereby the vectors will travel toand infect liver cells. The infected liver cells then will expressFactor VIII in therapeutic amounts. Factor VIII is not toxic to livercells and thus will continue to be expressed at therapeutic levels.

In yet another embodiment, when the DNA sequence encodes Factor IX or afragment, derivative, or analogue thereof, the promoter controlling theDNA sequence is preferably a strong promoter that is nottissue-specific, such as, for example, the Rous Sarcoma Virus promoter.Because it is believed that Factor IX is not toxic to most cells, theadenoviral vectors may be grown in any cell type, and may beadministered to a patient in an effective therapeutic amount, wherebythe adenoviral vectors will travel to and infect cells such as livercells, for example, whereby the Factor IX will be expressed intherapeutic amounts.

Several reports have revealed that, in transgenic mice, enhancedexpression of cDNA's can be obtained by the incorporation of 5' and 3'untranslated regions as well as introns (Choo et al. Nucl. Acids Res.,Vol. 15, pgs. 881-884 (1987); Brinster et al. PNAS, Vol. 85, pgs 836-840(1988); Jallat et al. EMBO J., Vol. 9, No. 10, pgs. 3295-3301 (1990);and Choi et al. Mol. Cell. Biol., Vol. 11, pgs. 3070-3074 (1991)). Theinclusion of genomic elements does not always result in improvedexpression. Furthermore, the effectiveness of genomic elements inimproving expression of exogenous genes incorporated into an adenoviralvector backbone has not been demonstrated previously.

In one embodiment, the DNA sequence encoding a clotting factor also mayinclude introns and other genomic elements to enhance expression. Theterm "genomic element," as used herein, means a sequence of nucleotidesin a naturally occurring gene that is not normally incorporated into thecDNA, and which is not part of the adenoviral genome. Such genomicelements which may be included in the vector include, but are notlimited to, introns, the 5' untranslated region, and the 3' untranslatedregion of the gene encoding the clotting factor, or portions of such 5'and 3' untranslated regions and introns. Examples of introns which maybe employed include, but are not limited to, any of the seven introns ofthe Factor IX gene, or portions thereof (EMBO J., Vol. 9, No. 10, pgs.3295-3301 (1990)); or any of the twenty-five introns of the Factor VIIIgene (Gitschier, Nature, 312:326-330 (1984)), or portions thereof; orthe first exon and intron of the apolipoprotein A-1 gene.

When the DNA sequence encodes Factor IX or a fragment, derivative, oranalogue thereof, the vector may, in one embodiment, further include thefull 3' untranslated region of the Factor IX DNA sequence. In anotherembodiment, the vector may further include the full 5' untranslatedregion and a centrally truncated first intron. In yet anotherembodiment, the vector may further include the full 3' untranslatedregion, the full 5' untranslated region, and a centrally truncated firstintron. Most preferably, the vector contains all of these elements. In afurther embodiment, the vector may further include the full 7th intronof the Factor IX gene.

When such elements are included in the vector, improved levels ofexpression of Factor IX are obtained. Although the scope of the presentinvention is not intended to be limited to any theoretical reasoning,such improved expression may be due to (i) the incorporation ofenhancers in the genomic sequences; (ii) stabilization of the mRNA;(iii) improved processing and transport of the mRNA to the cytoplasm;and/or (iv) improved polyadenylation.

In another embodiment, the first exon and first intron of theapolipoprotein A-1 gene may be employed, if desired, with theapolipoprotein A-1 gene promoter. (PNAS, Vol. 80, pgs. 6147-6151(October 1983); J. Biol. Chem., Vol. 266, No. 27, pgs. 18045-18050(September 1991)). The above-mentioned introns and/or exons also may beused in combination with the 5' untranslated region and/or the 3'untranslated region of the gene encoding the clotting factor.

In yet another embodiment, the above-mentioned introns and/or exonsand/or promoter of the apolipoprotein A-1 gene may be used incombination with the apolipoprotein A-1 5' untranslated region and/orthe apolipoprotein A-1 3' untranslated region and poly A signal. In oneembodiment, the above-mentioned introns and/or exons and/or promoter ofthe apolipoprotein A-1 gene are used in combination with theapolipoprotein A-1 3' untranslated region and poly A signal.

In a further embodiment, when the DNA sequence encodes Factor VIII or afragment, derivative, or analogue thereof, the above-mentioned intronsand/or exons, and/or promoter of the apolipoprotein A-1 gene may be usedin combination with the 5' untranslated region and/or 3' untranslatedregion and poly A signal of the human Factor IX gene. In one embodiment,the above-mentioned introns and/or exons and/or promoter of theapolipoprotein A-1 gene are used in combination with the 3' untranslatedregion and poly A signal of the human Factor IX gene.

In one preferred embodiment, the apolipoprotein A-1 promoter may beemployed, alone or in combination with the first exon and/or firstintron of the apolipoprotein A-1 gene, in combination with the FactorVIII gene.

The adenoviral vector which is employed may, in one embodiment, be anadenoviral vector which includes essentially the complete adenoviralgenome (Shenk et al., Curr. Top. Microbiol. Immunol., 111(3): 1-39(1984). Alternatively, the adenoviral vector may be a modifiedadenoviral vector in which at least a portion of the adenoviral genomehas been deleted.

In the preferred embodiment, the adenoviral vector comprises anadenoviral 5' ITR; an adenoviral 3' ITR; an adenoviral encapsidationsignal; at least one DNA sequence encoding a clotting factor; and apromoter controlling the at least one DNA sequence encoding a clottingfactor. The vector is free of at least the majority of adenoviral E1 andE3 DNA sequences, but is not free of all of the E2 and E4 DNA sequences,and DNA sequences encoding adenoviral proteins promoted by theadenoviral major late promoter.

In one embodiment, the vector also is free of at least a portion of atleast one DNA sequence selected from the group consisting of the E2 andE4 DNA sequences.

In another embodiment, the vector is free of at least the majority ofthe adenoviral E1 and E3 DNA sequences, and is free of a portion of theother of the E2 and E4 DNA sequences.

In still another embodiment, the gene in the E2a region that encodes the72 kilodalton binding protein is mutated to produce a temperaturesensitive protein that is active at 32° C., the temperature at which theviral particles are produced. This temperature sensitive mutant isdescribed in Ensinger et al., J. Virology, 10:328-339 (1972), Van derVliet et al., J. Virology, 15:348-354 (1975), and Friefeld et al.,Virology, 124:380-389 (1983).

Such a vector, in a preferred embodiment, is constructed first byconstructing, according to standard techniques, a shuttle plasmid whichcontains, beginning at the 5' end, the "critical left end elements,"which include an adenoviral 5' ITR, an adenoviral encapsidation signal,and an E1a enhancer sequence; a promoter (which may be an adenoviralpromoter or a foreign promoter); a multiple cloning site (which may beas hereinabove described); a poly A signal; and a DNA segment whichcorresponds to a segment of the adenoviral genome. The vector also maycontain a tripartite leader sequence. The DNA segment corresponding tothe adenoviral genome serves as a substrate for homologous recombinationwith a modified or mutated adenovirus, and such sequence may encompass,for example, a segment of the adenovirus 5 genome no longer than frombase 3329 to base 6246 of the genome. The plasmid may also include aselectable marker and an origin of replication. The origin ofreplication may be a bacterial origin of replication. Representativeexamples of such shuttle plasmids include pAVS6, shown in FIG. 5. Adesired DNA sequence encoding a clotting factor may then be insertedinto the multiple cloning site to produce a plasmid vector.

This construct is then used to produce an adenoviral vector. Homologousrecombination is effected with a modified or mutated adenovirus in whichat least the majority of the E1 and E3 adenoviral DNA sequences havebeen deleted. Such homologous recombination may be effected throughco-transfection of the plasmid vector and the modified adenovirus into ahelper cell line, such as 293 cells, by CaPO₄ precipitation. Upon suchhomologous recombination, a recombinant adenoviral vector is formed thatincludes DNA sequences derived from the shuttle plasmid between the NotI site and the homologous recombination fragment, and DNA derived fromthe E1 and E3 deleted adenovirus between the homologous recombinationfragment and the 3' ITR.

In one embodiment, the homologous recombination fragment overlaps withnucleotides 3329 to 6246 of the adenovirus 5 (ATCC VR-5) genome.

Through such homologous recombination, a vector is formed which includesan adenoviral 5' ITR, an adenoviral encapsidation signal; an E1aenhancer sequence; a promoter; at least one DNA sequence encoding aclotting factor; a poly A signal; adenoviral DNA free of at least themajority of the E1 and E3 adenoviral DNA sequences; and an adenoviral 3'ITR. The vector also may include a tripartite leader sequence. In oneembodiment, the tripartite leader sequence is deleted from theadenoviral vector, or the tripartite leader sequence contains one ormore mutations such that a polypeptide encoded by such tripartite leadersequence is not expressed. Applicants have found that, by deleting ormutating the tripartite leader sequence of the adenoviral vector, onemay obtain improved expression of the clotting factor. The vector maythen be transfected into a helper cell line, such as the 293 helper cellline (ATCC No. CRL1573), which will include the E1a and E1b DNAsequences, which are necessary for viral replication, and to generateinfectious adenoviral particles. Transfection may take place byelectroporation, calcium phosphate precipitation, microinjection, orthrough proteoliposomes.

The cloning vector hereinabove described may include a multiple cloningsite to facilitate the insertion of the at least one DNA sequenceencoding a clotting factor into the cloning vector. In general, themultiple cloning site includes "rare" restriction enzyme sites; i.e.,sites which are found in eukaryotic genes at a frequency of from aboutone in every 10,000 to about one in every 100,000 base pairs. Anappropriate vector in accordance with the present invention is thusformed by cutting the cloning vector by standard techniques atappropriate restriction sites in the multiple cloning site, and thenligating the DNA sequence encoding a clotting factor into the cloningvector.

The infectious viral particles (i.e., the adenoviral vector) aretransduced into eukaryotic cells, such as hepatocytes, whereby the atleast one DNA sequence encoding a clotting factor is expressed by theeukaryotic cells in a host.

The vector, consisting of infectious, but replication-defective, viralparticles, which contain at least one DNA sequence encoding a clottingfactor, is administered in an amount effective to treat hemophilia in ahost. In one embodiment, the vector particles may be administered in anamount of from 1 plaque forming unit (pfu) to about 10¹⁴ plaque formingunits, preferably from about 1×10⁶ plaque forming units to about 1×10¹³plaque forming units and more preferably from about 1×10⁸ plaque formingunits per kg to about 2×10¹⁰ plaque forming units per kg, and mostpreferably from about 1×10⁸ pfu/kg to about 1×10¹⁰ pfu/kg. The host maybe a human or non-human animal host. The preferred non-human animal hostis a mammal, most preferably a dog or a non-human primate.

Preferably, the infectious vector particles are administeredsystemically, such as, for example, by intravenous administration (suchas, for example, via peripheral vein injection) or administered via theportal vein, to the bile duct, intramuscularly, intraperitoneally, orintranasally.

The vector particles may be administered in combination with apharmaceutically acceptable carrier suitable for administration to apatient. The carrier may be a liquid carrier (for example, a salinesolution), or a solid carrier, such as, for example, mirocarrier beads.

As hereinabove stated, the inventors have found that the incorporationof genomic elements into the adenoviral vector provides for enhancedexpression of the DNA sequence encoding a clotting factor. Thus, inaccordance with another aspect of the present invention, there isprovided an adenoviral vector including at least one DNA sequenceencoding a heterologous protein, and at least one genomic elementaffecting the expression of such DNA sequence. The term "genomicelement" is used as previously defined. Such genomic elements include,but are not limited to, introns, the 5' untranslated region, and the 3'untranslated region, and portions of said introns and 3' and 5'untranslated regions. The adenoviral vector may be as hereinabovedescribed.

The DNA sequence encoding a heterologous protein may be a DNA sequencewhich encodes at least one therapeutic agent. The term "therapeutic" isused in a generic sense and includes treating agents, prophylacticagents, and replacement agents.

DNA sequences encoding therapeutic agents which may be placed into theadenoviral vector include, but are not limited to, DNA encoding FactorVIII and Factor IX as hereinabove described; DNA encoding cytokines; DNAsequences encoding tumor necrosis factor (TNF) genes, such as TNF-α;genes encoding interferons such as Interferon-α, Interferon-β, andInterferon-γ; genes encoding interleukins such as IL-1, IL-1β, andInterleukins 2 through 14; genes encoding GM-CSF; genes encodingadenosine deaminase, or ADA; genes which encode cellular growth factors,such as lymphokines, which are growth factors for lymphocytes; genesencoding soluble CD4; T-cell receptors; the LDL receptor, ApoE, ApoC,ApoAI and other genes involved in cholesterol transport and metabolism;the alpha-1 antitrypsin (α1AT) gene, the ornithine transcarbamylase(OTC) gene, the CFTR gene, the insulin gene, viral thymidine kinasegenes, such as the Herpes Simplex Virus thymidine kinase gene, thecytomegalovirus virus thymidine kinase gene, and the varicella-zostervirus thymidine kinase gene; Fc receptors for antigen-binding domains ofantibodies, and antisense sequences which inihibit viral replication,such as antisense sequences which inhibit replication of hepatitis B orhepatitis non-A non-B virus.

Promoters which control the DNA sequence may be selected from thosehereinabove described.

In one embodiment, the genomic element and the DNA sequence encoding aheterologous protein are part of the same endogenous gene. For example,the adenoviral vector may include DNA encoding Factor IX and a Factor IXgenomic element(s). In another embodiment, the DNA sequence encoding aheterologous protein and the genomic element are taken from differentendogenous genes. For example, the adenoviral vector may include DNAencoding Factor VIII and Factor IX genomic elements.

In yet another embodiment, an adenoviral vector may be constructedwherein the adenoviral vector includes DNA encoding a heterologousprotein and at least one genomic element(s) from the same endogenousgene. The DNA encoding a heterologous protein may be modified such thatat least one exon normally present in the DNA encoding the heterologousprotein is removed and replaced with one or more exons present inanother gene.

Although the scope of this aspect of the present invention is not to belimited to any theoretical reasoning, Applicants believe that, by theinclusion of at least one genomic element in an adenoviral vectorincluding at least one DNA sequence encoding a heterologous protein, oneis able to approximate endogenous transcription, RNA processing, andtranslation of the DNA sequence encoding a heterologous protein, therebyproviding for increased expression of the heterologous protein.

The invention will now be described with respect to the followingexamples; however, the scope of the present invention is not to belimited thereby.

EXAMPLE 1 Construction of an Adenoviral Vector Including a Factor IXGene

A. Construction of pG1H9

pG1 (FIG. 1), which is a retroviral plasmid vector including a 5' LTRderived from Moloney Sarcoma Virus, a multiple cloning site, and a 3'LTR from Moloney Murine Leukemia Virus, and is described in PCTApplication No. W091/10728, published Jul. 25, 1991, was cut with BamHIand HindIII. pLIXSN (Palmer et al, Blood, Vol. 73, No. 2, pgs. 438-445(February 1989)), which contains a Factor IX gene, an SV40 promoter, anda neo^(R) gene, was also cut with BamHI and HindIII. The resultingBamHI-HindIII fragment, which contains the Factor IX gene, was thenligated to the BamHI-HindIII digested pG1 to form pG1H9. (FIG. 2). TheFactor IX gene could also have been obtained according to the proceduresdisclosed in U.S. Pat. No. 4,994,371.

B. Construction of pG1H9B

pG1H9B (FIG. 4) was constructed so that the 5' portion of the humanFactor IX cDNA starting at the first ATG is identical to the natural 5'human Factor IX sequence. Such is not the case for pG1H9 because of aninversion in the DNA sequence.

pG1H9B was constructed as follows. First, a cDNA clone of human FactorIX was generated by PCR amplification of human liver cDNA followed bysubcloning into the plasmid pBluescript SK- (Stratagene, La Jolla,Calif.). The resulting plasmid was designated phfacIX (FIG. 3). The 5'end of the Factor IX sequence in this plasmid was then used to replacethe 5' end of the Factor IX sequence in G1H9. phfacIX then was cut withBamHI and DraI, and the 334 bp fragment corresponding to the 5' end ofthe Factor IX cDNA was isolated. pG1H9 was cut with DraI and ClaI andthe 1253 bp fragment encoding the 3' end of the Factor IX cDNA wasisolated. The two isolated DNA fragments encoding Factor IX cDNA wereligated into the pG1H9 backbone which had been cut with BamHI and ClaIto generate pG1H9B (FIG. 4).

C. Construction of pAVS6

The adenoviral construction shuttle plasmid pAvS6 (FIG. 5, and alsodescribed in PCT Application Nos. W094/23582, published Oct. 27, 1994,and W095/09654, published Apr. 13, 1995), was constructed in severalsteps using standard cloning techniques including polymerase chainreaction based cloning techniques. First, the 2913 bp BglII, HindIIIfragment was removed from Ad-dl327 and inserted as a blunt fragment intothe XhoI site of pBluescript II KS- (Stratagene, La Jolla, Calif.).

Ad-dl327 is identical to adenovirus 5 except that an XbaI fragmentincluding bases 28591 to 30474 (or map units 78.5 to 84.7) of theAdenovirus 5 genome, and which is located in the E3 region, has beendeleted. The E3 deletion in Ad-dl327 is similar to the E3 deletion inAd-dl324, which is described in Thimmapaya et al., Cell, Vol. 31, pg.543 (1983). The complete Adenovirus 5 genome is registered as Genbankaccession #M73260, incorporated herein by reference, and the virus isavailable from the American Type Culture Collection, Rockville, Md.,U.S.A. under accession number VR-5.

Ad-dl327 was constructed by routine methods from Adenovirus 5 (Ad5). Themethod is outlined briefly as follows and previously described by Jonesand Shenk, Cell 13:181-188, (1978). Ad5 DNA is isolated by proteolyticdigestion of the virion and partially cleaved with Xba 1 restrictionendonuclease. The Xba 1 fragments are then reassembled by ligation as amixture of fragments. This results in some ligated genomes with asequence similar to Ad5, except excluding sequences 28591 bp to 30474bp. This DNA is then transfected into suitable cells (e.g. KB cells,HeLa cells, 293 cells) and overlaid with soft agar to allow plaqueformation. Individual plaques are then isolated, amplified, and screenedfor the absence of the 1878 bp E3 region Xba 1 fragment.

The orientation of this fragment was such that the BglII site wasnearest the T7 RNA polymerase site of pBluescrpt II KS⁻ and the HindIIIsite was nearest the T3 RNA polymerase site of pBluescript II KS⁻. Thisplasmid was designated pHR.

Second, the ITR, encapsidation signal, Rous Sarcoma Virus promoter, theadenoviral tripartite leader (TPL) sequence and linking sequences wereassembled as a block using PCR amplification. The ITR and encapsidationsignal (sequences 1-392 of Ad-dl327 identical to sequences from Ad5,Genbank accession #M73260! incorporated herein by reference) wereamplified (amplification 1) together from Ad-dl327 using primerscontaining NotI or AscI restriction sites. The Rous Sarcoma Virus LTRpromoter was amplified (amplification 2) from the plasmid pRC/RSV(sequences 209 to 605; Invitrogen, San Diego, Calif.) using primerscontaining an AscI site and an SfiI site. DNA products fromamplifications 1 and 2 were joined using the "overlap" PCR method(amplification 3) (Horton et al., BioTechniques, 8:528-535 (1990)) withonly the NotI primer and the SfiI primer. Complementarity between theAscI containing end of each initial DNA amplification product fromreactions 1 and 2 allowed joining of these two pieces duringamplification. Next the TPL was amplified (amplification 4) (sequences6049 to 9730 of Ad-dl327 identical to similar sequences from Ad5,Genbank accession #M73260!) from cDNA made from mRNA isolated from 293cells (ATCC Accession No. CRL 1573) infected for 16 hrs. with Ad-dl327using primers containing SfiI and XbaI sites respectively. DNA fragmentsfrom amplification reactions 3 and 4 were then joined using PCR(amplification 5) with the NotI and XbaI primers, thus creating thecomplete gene block.

Third, the ITR-encapsidation signal-TPL fragment was then purified,cleaved with NotI and XbaI and inserted into the NotI, XbaI cleaved pHRplasmid. This plasmid was designated pAvS6A⁻ and the orientation wassuch that the NotI site of the fragment was next to the T7 RNApolymerase site.

Fourth, the SV40 early polyA signal was removed from SV40 DNA as anHpaI-BamHI fragment, treated with T4 DNA polymerase and inserted intothe SalI site of the plasmid pAvS6A- to create pAvS6 (FIG. 5).

D. Construction of Av1H9B

Factor IX cDNA (FIG. 6), which contains the entire protein codingsequence, 26 base pairs of 5' untranslated DNA (assuming translationstarts at the third ATG of the message) and 160 base pairs of 3'untranslated DNA, was excised from pG1H9B by restriction digestion withClaI, followed by filling in the 5' overhang using Klenow, followed byrestriction digestion with SmaI. The Factor IX cDNA could also have beenobtained according to the procedures disclosed in U.S. Pat. No.4,994,371.

The fragment encoding Factor IX was isolated by electrophoresis in a1.0% agarose gel followed by electroelution of the DNA. This fragmentwas subcloned into pAvS6 which had been linearized with EcoRV andtreated with calf intestinal phosphatase. The resulting shuttle plasmidpAvS6H9B (FIG. 7), contains the 5' inverted terminal repeat ofadenovirus type 5 (Ad 5), the origin of replication of Ad 5, the Ad 5encapsidation signal, the E1a enhancer, the RSV promoter, the tripartiteleader sequence of Ad 5, Factor IX cDNA, the SV40 early polyadenylationsignal, and Ad 5 sequences from nucleotide positions 3329-6246.

The recombinant adenoviral vector Av1H9B was generated as depicted inFIG. 8. 1.5×10⁶ 293 cells were cotransfected in a 60 mm tissue culturedish with 4 μg of the large Cla I fragment of Ad-dl327 (an E3 deletionmutant of Ad 5) and 5 μg of shuttle plasmid pAvS6H9B digested with Not Iand Kpn I. Transfections were done using BRL's Transfinity calciumphosphate transfection system. Approximately 15 hours aftertransfection, medium containing DNA/calcium phosphate precipitate wasremoved from the dishes, the cells were gently washed with PBS, thenoverlaid with a 1:1 mixture of 2× MEM (GIBCO's 2× Modified Eagle Mediumsupplemented with 15% FBS) and 2% SeaPlaque agarose.

Plaques were picked using sterile Pasteur pipettes and transferred into0.1 ml of infection medium (Improved Minimum Essential Medium (IMEM), 1%FBS) in an Eppendorf tube. Resuspended plaques were subjected to threefreeze/thaw cycles, then cleared of cell debris by a 15 secondcentrifugation at full speed in a microfuge.

Recombinant adenovirus was amplified in 293 cells as follows.Approximately 5×10⁵ 293 cells per dish were seeded into 30 mm dishes.The next day medium was removed from the cells and replaced with 0.2 mlof infection medium and 0.1 ml of a resuspended plaque. The plates wereincubated with gentle rocking for 90 minutes in a 37° C., 5% CO₂incubator. Subsequently, 2 ml of complete medium (IMEM, 10% FBS) wereadded. Approximately 40 hours later a cytopathic effect was clearlyvisible; cells were rounded-up and beginning to detach from the plate.Cells and medium were transferred to plastic tubes, subjected to fourfreeze/thaw cycles, and centrifuged at 2000×g for 5 minutes. Theresulting supernatant is referred to as the crude viral lysate (CVL1).

Viral DNA was isolated from an aliquot of each CVL1, then analyzed forthe presence of Factor IX cDNA by PCR, as follows. A 60 μl aliquot ofsupernatant was transferred into an Eppendorf tube and incubated at 80°C. for 5 minutes. The sample was centrifuged at full speed for 5 minutesin a microfuge, then 5 μl of the supernatant were used for PCR analysis.PCR analysis was done using the Perkin Elmer Cetus GeneAmp kit. Twodifferent pairs of primers which amplify different portions of the humanFactor IX cDNA were used. All samples yielded the expected amplifiedband.

EXAMPLE 2 In Vitro and In Vivo Function of the Vector of Example 1

Recombinant adenovirus vectors containing Factor IX cDNA were tested fortheir ability to express human Factor IX in 293 cells. Approximately5×10⁵ 293 cells were seeded per 60 mm dish. The next day, medium wasreplaced with 0.1 ml of recombinant adenovirus and 0.1 ml of infectionmedium. Plates were incubated for 1 hour with gentle rocking at 37° C.in 5% CO₂, followed by addition of 4 ml of complete medium. The cellswere gently washed five times with PBS, then 4 ml of complete mediumwere added. Media samples were collected 24 hours later and centrifugedat 1500×g for 5 minutes. Supernatants were assayed for human Factor IXby ELISA (Asserachrom IX:Ag ELISA kit, American Bioproducts), and thelevels were 445 and 538 ng/ml for the two samples, demonstrating thatthe recombinant adenoviral vectors are able to express human Factor IX.Uninfected 293 cells yielded background levels of Factor IX.

One recombinant adenovirus was selected for a large scale viruspreparation. Approximately 5×10⁶ 293 cells were seeded onto a 15 cmtissue culture dish. The next day, the medium was replaced with 4 ml ofinfection medium plus 1 ml of the crude viral stock. Then the plateswere incubated at 37° C., 5% CO₂ with gentle rocking for 90 minutes,followed by addition of 15 ml of complete medium. Approximately 40 hourslater, when a cytopathic effect was clearly visible, cells and mediumwere transferred to a 50 ml plastic tube. Cells were lysed by fivefreeze/thaw cycles and cell debris was removed by centrifugation at1500×g for five minutes. This supernatant was termed CVL2.

15 ml of CVL2 then was mixed with 35 ml of infection medium and 5 ml ofthis mixture was added to each of ten 15 cm plates of nearly confluent293 cells. The plates were incubated at 37° C., 5% CO₂ with gentlerocking for 1 hour, followed by addition of 15 ml complete medium toeach plate. Twenty-four hours later a cytopathic effect was observed;cells were rounded up, but not lysed. Cells and medium were centrifugedat 2000×g for 10 minutes. The cell pellet was resuspended in 6 ml ofcomplete medium. Cells were lysed by five freeze/thaw cycles, followedby centrifugation in a SW40 rotor at 7000 rpm for 10 minutes at 4° C.Virus in the supernatant was purified on a CsCl step gradient asfollows. 3.0 ml of 1.25 g/ml CsCl in TD buffer (25 mM Tris, 137 mM NaCl,5 mM KCl, 0.7 mM Na₂ HPO₄, pH7.5) was placed in an ultraclear Beckman#344060 ultracentrifuge tube. This was underlaid with 3.0 ml of 1.40g/ml CsCl in TD buffer. The CsCl layers were overlaid with 4.5 ml ofviral supernatant. Centrifugation was done at 35,000 rpm, 22° C. for 1hour in a SW40 rotor. Two bands were visible, an upper band thatconsists of empty capsids and a lower band consisting of intactrecombinant adenovirus.

The lower band was collected with a 3 ml syringe and a 21 gauge needle,and then rebanded as follows. 9.0 ml of 1.33 g/ml CsCl in TD buffer wasplaced into an ultracentrifuge tube. This was overlaid with the viruscollected from the first spin. Centrifugation was done at 35,000 rpm,22° C. for 18 hours. The opalescent band was collected as above andglycerol was added to a final concentration of 10%. The adenovirus wasdialyzed against one liter of 10 mM Tris pH 7.4, 10 mM MgCl₂, and 10%glycerol at 4° C. Dialysis was done for 4 hours and the buffer waschanged three times at one hour intervals. The virus was recovered andstored at -70° C. in aliquots in sterile Eppendorf tubes. The titer ofthis virus preparation was 9.6×10⁹ pfu/ml.

In the first in vivo experiment, the recombinant adenovirus Av1H9B wasinjected into three C57BL/6 mice by three different methods: anintraparenchymal injection into the liver, infusion into the portalvein, and infusion into the tail vein.

The animal which received an intraparenchymal injection was anesthetizedunder Metofane. A longitudinal incision approximately 7 mm in length wasmade just below the xiphoid. Pressure was applied to the flanks causingprotrusion of the median and left lateral lobes. For injection, 0.1 mlof virus (1×10⁹ pfu) was diluted to 1.0 ml with Hanks Balanced SaltSolution (HBSS). The virus was injected into 4 different sites of theliver: 0.25 ml was injected into each half of the median lobe and intothe left and right sides of the left lateral lobe. Each injection wasdone over approximately one minute. Upon removal of the needle,hemostasis was achieved by placing small pieces of gelfoam over theinjection site. After 2 minutes, the gelfoam was removed, the liver wasgently placed into the abdominal cavity, and the skin incision wasclosed with autoclips. Animals awakened within several minutes ofsurgery and were ambulatory within one hour.

The animal which received a portal vein infusion of AdH9B wasanesthetized under Metofane. A midline longitudinal incision was madefrom the xiphoid to just above the pelvis. The intestines were gentlyexternalized to the left side of the animal with wet cotton tipapplicators. An 0.1 ml aliquot of virus (1×10⁹ pfu) was diluted to 1.0ml with HBSS. The viral suspension was infused over 30 seconds into theportal vein using a 1 ml syringe and a 27 gauge needle. A 3×3 mm pieceof gelfoam was placed over the injection site. The needle then waswithdrawn. Hemostasis was achieved by applying mild pressure to thegelfoam for 5 minutes using a wet cotton tip applicator. The gelfoam wasleft in place. The intestines were gently returned to the abdominalcavity. The incision was closed using autoclips. The animal awakenedwithin 30 minutes of surgery and was ambulatory within 1 hour.

A tail vein infusion of Av1H9B was performed using 0.1 ml of virus(1×10⁹ pfu) diluted to 1.0 ml with HBSS. The viral suspension wasinfused over a ten second period using a 27 gauge needle.

The animals which received an intraparenchymal injection and portal veininfusion, as well as a control mouse which received no virus, were bledvia the retro-orbital plexus on days 2, 6, and 9 after virus delivery.The animal which received a tail vein infusion was bled 2 days aftervirus delivery. Plasma levels of human Factor IX were determined byELISA. The results are shown in FIG. 9.

At this point, it was important to determine vector levels in the liversof the mice. Therefore, the animals which received an intraparenchymalinjection and a portal vein infusion and the negative control mouse weresacrificed on day 9 after infusion and the mouse which received a tailvein injection was sacrificed on day 2 after infusion. The liver of eachmouse was removed and extensively minced with a razor blade. One-half ofeach liver was placed into a 15 ml conical tube and 1.0 ml of lysisbuffer (10 mM Tris, 0.14 M NaCl, pH 8.6) was added. The tissue washomogenized using a 1 ml syringe and a 20 gauge needle. Next, 1.0 ml of2× PK buffer (200 mM Tris pH 7.5, 25 mM EDTA, 300 mM NaCl, 2% (w/v) SDS,and 500 μg/ml proteinase K) was added. The tube was inverted severaltimes, then incubated at 37° C. overnight. The samples were extractedtwice with phenol/chloroform (1:1) and once with chloroform/isoamylalcohol (24:1). DNA was ethanol precipitated, washed with 70% ethanol,and resuspended in 10 mM Tris, pH 7.5, 1 mM EDTA.

A Southern analysis was performed to quantitate the levels of vector inthe liver. Ten micrograms of each DNA sample were cut with BamHI. Thedigested DNA samples were subjected to electrophoresis in an 0.8% Seakemagarose gel in 40 mM Tris, 20 mM NaAcetate, 1 mM EDTA, pH7.5.

After electrophoresis, the gel was treated with 0.2 N NaOH, 0.6 M NaClfor 1 hour, then neutralized with 1 M Tris pH 7.4, 0.6 M NaCl for 30minutes. The DNA was transferred to a nylon membrane by blotting in10×SSC. The nylon membrane was baked at 80° C. for 1 hour in a vacuumoven. It was prehybridized for 3 hours at 42° C. in 5× Denhardt's, 5×SSC, 50 mM NaPhosphate pH 6.5, 250 μg/ml salmon sperm DNA, 0.1% SDS, and50% formamide. The membrane was hybridized for 24 hours at 42° C. in 1×Denhardt's, 5× SSC, 20 mM NaPhosphate pH 6.5, 100 g/ml salmon sperm DNA,0.1% SDS, 50% formamide, and 33 μCi of random primer labeled humanFactor IX cDNA. Random primer labeling was performed using the BRL kit.The membrane was washed in 2× SSC, 0.1% SDS for 20 minutes at roomtemperature, followed by a 30 minute wash in 2× SSC, 0.1% SDS at 50° C.,and then a 30 minute wash in 0.1× SSC, 0.1% SDS at 68° C. The membranewas exposed to film for 16 hours, then developed. A copy of theautoradiogram is shown in FIG. 10. All three routes of administrationyielded the same results. The Factor IX bands were the appropriate sizewith an intensity that indicated an average of 5-10 copies per livercell.

EXAMPLE 3 In Vivo Expression of Factor IX in Mice Injected with Av1H9B

A second large scale virus preparation of Av1H9B was performed using thesame protocol described above, except that 28 15 cm plates of 293 cellswere used to amplify the virus. This preparation yielded a much thickeropalescent band upon CsCl gradient centrifugation than the first viruspreparation. The titer of this virus preparation was 1.1×10¹¹ pfu/ml.

A second in vivo experiment, designed to follow the time course ofexpression, was initiated using the new Av1H9B preparation. Virus wasadministered to mice as described above, except that 0.1 ml of a virussuspension (1×10¹⁰ pfu) was diluted to 1.0 ml with infection medium.Twenty-seven mice received an injection of recombinant adenovirus: 20mice received a tail vein injection, 18 with Av1H9B and 2 with Av1lacZ4(encoding β-galactosidase), 4 mice received an intraparenchymalinjection of the liver, and 3 received an intramuscular injection. Anegative control mouse was not injected. The animals were bled once aweek for seven weeks. Plasma levels of human Factor IX are shown inTable I.

As shown in Table I, IP means intraparenchymal injection of Av1H9B, TVmeans a tail vein injection of Av1H9B, IM means an intramuscularinjection of Av1H9B, LacZ means a tail vein injection of Av1lacZ4, andNI means no injection (control).

                  TABLE I    ______________________________________    ng/ml Factor IX in plasma    Mouse  Injection Week 1   Week 2 Week 3 Week 4    ______________________________________     1.    TV        376      475    281    171     2.    TV        270      500    392    336     3.    TV        229      374    --     --     4.    TV        240      --     --     --     5.    TV        362      --     --     --     6.    TV        346      --     --     --     7.    TV        303      422    252    142     8.    TV        260      573    394    220     9.    TV        353      376    273    149    10.    TV        321      357    270    246    11.    TV        431      482    233    203    12.    TV        347      332    --     --    13.    TV        135      244    126    61    14.    TV        261      294    187    148    15.    TV        212      269    132    91    16.    TV        207      255    214    176    17.    TV        278      218    151    149    18.    TV        170      308    --     --    19.    IM        0.9      3.0    0.0    0.0    20.    IM        1.0      2.7    0.0    0.0    21.    IM        1.1      2.6    1.0    1.2    22.    IP        364      316    174    131    23.    IP        211      308    134    66    24.    IP        305      252    155    206    25.    IP        527      406    133    94    26.    LacZ      0.0      2.8    1.5    0.4    27.    LacZ      0.0      2.6    2.0    1.2    28.    NI        0.0      2.6    1.5    0.5    ______________________________________              Mouse Injection                             Week 5  Week 6                                           Week 7    ______________________________________               1.   TV       98      34    16               2.   TV       187     67    16               3.   TV       --      --    --               4.   TV       --      --    --               5.   TV       --      --    --               6.   TV       --      --    --               7.   TV       --      --    --               8.   TV       197     60    25               9.   TV       131     90    46              10.   TV       179     76    16              11.   TV       --      --    --              12.   TV       --      --    --              13.   TV       84      62    17              14.   TV       133     65    26              15.   TV       94      64    33              16.   TV       --      --    --              17.   TV       92      57    23              18.   TV       --      --    --              19.   IM       0.0     2.0   0.0              20.   IM       0.0     2.0   4.1              21.   IM       0.1     2.0   0.0              22.   IP       112     53    48              23.   IP       42      28    19              24.   IP       299     203   154              25.   IP       57      21    13              26.   LacZ     0.0     1.5   5.0              27.   LacZ     0.0     1.5   4.0              28.   NI       1.4     1.7   0.5    ______________________________________

EXAMPLE 4 Assay for Biological Activity of Human Factor IX

The biological activity of human Factor IX in mouse plasma wasdetermined by using an immunocapture, chromogenic assay. A 96-wellmicrotiter plate was coated with a BGIX1 monoclonal antibody obtainedfrom Elcatech, Inc., which recognizes, but does not inactivate, humanFactor IX. Coating was done by adding 100 μl of a 10 μg/ml suspension ofthe antibody to each well and incubating at room temperature overnight.Plasma samples obtained from Mouse 7 in Example 3 two weeks afterinjection with the recombinant adenovirus (100 μl of 1:5 and 1:10dilutions) were added to the wells and human Factor IX was allowed tobind. The wells were washed to remove unbound material, and capturedhuman Factor IX was activated by adding 100 μl of a 2 μg/ml suspensionof Factor XIa (Enzyme Research Labs) and incubating at 37° C. for 30minutes. The wells were washed, and then 100 μl of a mixture containing5.0 μg phospholipid (Kabi Pharmacia, Franklin, Ohio), 0.1 unit Factor X(Kabi), 0.5 unit Factor VIII (Elcatech), 3.4 μg I-2581 thrombininhibitor (Kabi) and 2.5 mM CaCl₂ were added. The plate was incubated at37° C. for 30 minutes, during which time Factor X was converted toFactor Xa. 100 μl of 0.5 mMN-N-alpha-benzyloxycarbonyl-D-arginyl-L-glycyl-L-arginine-p-nitroanilide-dihydrocholoride, a chromogenic Factor Xa substrate, then was added and theplate was incubated at room temperature for ten minutes. The colordevelopment was stopped by adding 50 μl of 50% acetic acid. Theabsorbance at 405 nm was determined using a Bio-Rad microplate reader.Standard curves (log-log and linear-linear) were generated using normalpooled human plasma, assuming Factor IX levels of 5000 ng/ml.Biologically active Factor IX was determined to be 51 ng/ml according tothe log-log method, and 415 ng/ml according to the linear-linear method.Such results are within experimental error, and indicate thatessentially all of the total Factor IX antigen determined in Example 3(422 ng/ml) is biologically active.

EXAMPLE 5 Construction of Adenoviral Vector Including DNA Encoding aFactor VIII Derivative

pAVS6H81 (FIG. 13) was constructed from pMT2LA (FIG. 11) and pAVS6.(FIG. 5). pMT2LA (Genetics Institute, Cambridge, Mass.) includes cDNAencoding a derivative of human Factor VIII in which the B domain ofFactor VIII is deleted. Such cDNA is further described in Toole et al.,Nature, Vol. 312, pgs. 342-349 (November 1984), Vehar et al., Nature,Vol. 312, pgs. 337-342 (November 1984), and Toole et al., PNAS, Vol. 83,pgs. 5939-5942 (August 1986). The cDNA is controlled by a Rous SarcomaVirus promoter. The 4.6 kb cDNA (FIG. 12) contains no natural 5'untranslated DNA, and 216 bp of 3' untranslated DNA. The B domaindeletion removes nucleotides 2334-4973 of the coding sequence of thefull length Factor VIII. The cDNA for B domain deleted Factor VIII couldalso have been obtained according to the procedures disclosed in U.S.Pat. No. 4,868,112.

The cDNA was excised from the plasmid pMT2LA by restriction digestionwith XhoI and SalI. The ends were filled in using Klenow, and thefragment encoding the Factor VIII derivative was isolated on an 0.8%agarose gel, followed by electroelution. This fragment was subclonedinto the EcoRV site of pAVS6 (FIG. 10) to generate pAVS6H81. (FIG. 13.)

The recombinant adenoviral vector Av1H81 is generated as depicted inFIG. 14. 1.5×10⁶ 293 cells are cotransfected in a 60 mm tissue culturedish with 4 μg of the large ClaI fragment of Ad dl 327 and 5 μg ofpAvS6H81 digested with NotI. Transfections are done using BRL'sTransfinity calcium phosphate transfection system. Approximately 15hours after transfection, medium containing DNA/calcium phosphateprecipitate is removed from the dishes, the cells are gently washed withPBS, then overlaid with a mixture of 2× MEM and 2% Sea Plaque agarose.

Recombinant adenovirus can be prepared from plaques and analyzed by PCRfor the presence of human Factor VIII cDNA.

EXAMPLE 6 Generation of Adenoviral Vectors Including DNA Encoding FactorVIII Plus Genomic Elements

A. Construction of pAvALH81

A schematic of the construction of pAvALH81 is shown in FIG. 15. Themouse albumin promoter (Zaret et al., Proc. Nat. Acad. Sci. USA, Vol.85, pgs. 9076-9080 (1988)), containing 3.5 copies of a liver-specifictranscription factor binding site (eG binding sites, Liu et. al., Mol.Cell. Biol., Vol. 11, pgs. 773-784 (1991) and Di Persio et al., Mol.Cell. Biol., Vol. 11, pgs. 4405-4414 (1991)) was PCR amplified frompAT2-3eG (FIG. 15, provided by Kenneth Zaret, Brown University,Providence, R.I.) using oligo MGM8.293,

    5'-GGC TAG ACG CGT GCT ATG ACC ATG ATT ACG AA-3'           (SEQ ID NO:1)

complementary to nts 4281-4299 of pAT2-3eG with the addition of an MluIrestriction site, as the 5' oligo, and oligo MGM5.293,

    5'-GGT ACG GAT CCA TCG ATG TCG ACG CCG GAA AGG TGA TCT GTGT-3'(SEQ ID NO:2)

complementary to nts 5231-5212 of pAT2-3eG with the addition of BamHI,ClaI, and SalI restriction sites, as the 3' oligo. The PCR product wascut with MluI and BamHI and inserted into pAVS6 (FIG. 5) cut with MluIand BamHI to generate pAVAL1 (FIG. 15). The sequence of the 964 bpPCR-generated albumin promoter has been verified by sequencing. Inaddition, at least 50 bp on either side of the MluI site (nt 428) andBamHI site (nt 1392) in pAVAL1 (FIG. 15) have also been verified bysequencing. The plasmid pAT-2-3eG is prepared according to theprocedures disclosed in DiPersio et al., Mol. Cell. Biol., 11:4405-4414(1991) and Zaret et al., Proc. Nat. Acad. Sci., Vol. 85, pgs. 9076-9080(1988), which disclose the preparation of a mouse albumin promoter withtwo copies of a liver-specific transcription factor binding site. Theplasmid pAT2-3eG has been deposited under the Budapest Treaty in theAmerican Type Culture Collection, 1230 Parklawn Drive, Rockville, Md.20892, and assigned accession number 69603.

The ITR, encapsidation signal (see construction of pAVS6) and thealbumin promoter were removed from pAVAL1 by digestion with NotI (theends were filled in with T4 DNA polymerase) and SalI, and inserted intopGEM(sac) (FIG. 15), cut with SalI and SmaI to generate pGEMalb (FIG.15) (pGEM(sac) was created by cutting pGEM (FIG. 15, Promega; Madison,Wis.) with SacI, and blunting the ends with T4 DNA polymerase andreligation, thereby removing the SacI site.) A 1914 bp fragment,containing the 5' region of the B-domain deleted factor VIII cDNA wasisolated from pMT2LA (FIG. 11) by digestion with BamHI (filling in the5' end with T4 DNA polymerase) and digestion with XhoI, and insertedinto pGEMalb digested with HindIII (filled in with T4 DNA polymerase)and SalI, to generate pGEMalbF8B (FIG. 15). pGEMalbF8B was cut with MluIand SpeI, and the resulting 1556 bp fragment was inserted into pAvS6H81(FIG. 13), cut with MluI and SpeI, to generate the adenovirus shuttleplasmid, pAvALH81 (FIG. 16). At least 50 bp on either side of the MluIsite (nt 429) and SpeI site (nt 1985) have been verified by sequencingof Av1ALH81 viral DNA (see below). The sequence of the Factor VIIIB-domain deleted cDNA has been verified by sequencing of bases 1075 to5732 from the original plasmid, pMT2LA (FIG. 11) obtained from GeneticsInstitute. It should be noted that this sequence differs from thesequence reported by Genetics Institute by two bases. One base change,nt 1317 of pMT2LA was reported by Genetics Institute to be a T (Tooleet. al., Nature, Vol. 312, pgs. 342-347 (1984) and by Wood et. al.,Nature, Vol. 312, pgs. 330-337 (1984) to be an A. In addition, nt 5721of pMT2LA, reported by Genetics Institute to be a T, was deleted, thuscreating a BamHI site within the Factor VIII 3' untranslated region.This mutation does not change the Factor VIII coding region.

B. Construction of pAvAPH81

A schematic of the construction of pAvAPH81 is shown in FIG. 17. A 1913bp fragment was isolated from pAVS6H81 (FIG. 13) by digestion with BamHI, and inserted into pGEM(sac) (FIG. 15) cut with Bam HI, to createpGemF8B2 (FIG. 17). The ApoA1 promoter, first exon (untranslated), firstintron, and second exon to the ATG (Genbank #X07496) were PCR amplifiedusing pBGS19-AIgI (FIG. 17) as the template. pBGS19-AIgI (FIG. 17) wasconstructed in two steps: 1) The 13 kb SalI fragment was removed fromLambda Al 103 (Swanson, et. al., Transgenic Research, Vol. 1, pgs.142-147 (1992), and inserted into pUC19 (FIG. 17, Gibco BRL) to generatepUC19-AIgI (FIG. 17). 2) The 2 kb SmaI fragment was isolated frompUC19-AIgI (FIG. 30) and inserted into pBGS19 (FIG. 17) to generatepBGS19-A1gI (FIG. 17). pBGS19 (ATCC No. 37437) is a kanamycin analog ofpUC19. PCR-amplification of pBGS19-AIgI was performed using oligoSSC1.593,

    5'GCT CTA GAA CGC GTC GGT ACC CGG GAG ACC TGC AAG CC-3'    (SEQ ID NO:3)

complementary to bases 5862 to 16 of pBGS19-AIgI, containing an XbaI anda MluI site, as the 5' oligo, and a 3' oligo SSC2.593,

    5'-GGA ATT CGA GCT CTAT TTG CAT CCT GAA GGG CCG TGG GGA CCT GG-3'(SEQ ID NO:4)

complementary to human factor VIII (Genbank #KO1740, nts 151-165 (to theSacI site), and nts 463-487 of pBGS19-AIgI, complementary to the ApoA1gene (Genbank #X07496) with the addition of a SacI and an EcoRI site.The PCR fragment was digested with XbaI and SacI and the resulting 509bp fragment was inserted into pGemF8B2 (FIG. 17) digested withXbaI-SacI, to generate pGemAPF8B (FIG. 17). pGemAPF8B was then digestedwith MluI-SpeI, and the resulting 1084 bp fragment was ligated intopAVS6H81 (FIG. 18) cut with MluI and SpeI, to generate the shuttleplasmid, pAvAPH81 (FIG. 18). The sequence of pAvAPH81, from nts 290 to1619, which include the PCR-generated ApoA1 promoter region, and allcloning junctions, has been verified.

C. Construction of pAvALAPH81

A schematic of the construction of pAvALAPH81 is shown in FIG. 19. ASalI site was added upstream from the ApoA1 transcription initiationsite by PCR amplification of pGemAPF8B (FIG. 17) using a 5' oligoSSC3.593,

    5'-GAA TTC GTC GAC AGA GAC TGC GAG AAG GAG GTG CG-3'       (SEQ ID NO:5)

complementary to the ApoA1 gene (Genebank #X07496) and nts 252-274 ofpBGS19-A1g1 (FIG. 17) with the addition of an EcoRI and a SalI site, anda 3' oligo, SSC2.593 (see above). The PCR fragment was digested withSalI-SacI, and the resulting 250 bp fragment was inserted into pGemF8B2(FIG. 17) cut with SalI-SacI, to create pGemAPexF8B (FIG. 19). Theplasmid, pALAPF8B (FIG. 19) was generated by a 3-way ligation of the 953bp MluI-SalI fragment isolated from pGEMalb (FIG. 15), the 825 bpSalI-SpeI fragment isolated from pGemAPexF8B (FIG. 19), inserted intopGemAPF8B (FIG. 17) cut with MluI-SpeI. The 1778 bp MluI-SpeI fragmentwas isolated from pALAPF8B (FIG. 19) and inserted into pAVS6H81 (FIG.13) to generate the shuttle plasmid, pAvALAPH81 (FIG. 20).

D. Generation of Recombinant Adenovirus Vectors

The recombinant adenoviral vector, Av1ALH81, was generated as outlinedin FIG. 21. 2×10⁶ 293 cells were cotransfected in a 100 mm tissueculture dish with 10 μg of the large ClaI fragment of Ad-dl327, and 10μg of the undigested shuttle plasmid, pAvALH81 (FIG. 16). Transfectionswere performed using the Transfinity calcium phosphate transfectionsystem from BRL. Approximately 12 hrs after DNA addition, the cells werewashed 2× with 1× PBS, then overlaid with a 1:1 mixture of 2× MEM(GIBCO'S 2× Modified Eagle Medium supplemented with 15% FBS) and 2%SeaPlaque agarose.

Plaques were harvested with sterile Pasteur pipettes and transferredinto 0.1 ml of infection medium (Improved Minimum Essential Medium (IMEM!, 2% FBS) in an Eppendorf tube, and subjected to three rounds offreeze/thaw cycles. Cell debris was removed by a 15 secondcentrifugation at full speed in a microfuge.

Plaques were screened for the presence of recombinant adenovirus asfollows. Approximately 5×10⁵ 293 cells were seeded per well of 6-welltissue culture plates. The following day, media was removed from thecells and replaced with 0.4 ml of infection medium and 0.05 ml of theresuspended plaque. The plates were incubated with rocking, for 90 min.in a 37° C./5% CO₂ incubator, after which 2 ml of complete medium (IMEM,10% FBS) were added. When the cytopathic effect (CPE) was complete,cells were rounded and becoming detached from the plate (approximately40-120 hrs after infection), cells and medium were transferred to 15 mlconical tubes, and centrifuged at 1000 rpm for 5 min. to pellet cells.The medium was removed from the cell pellet, and the cells wereprocessed as follows.

Cells were resuspended in 250 μl of PK buffer (5 mM Tris pH 8.0, 5 mMEDTA, pH 8.0, and 0.5% SDS) plus 250 μl of Proteinase K (1 mg/ml), andincubated 4 hrs or overnight at 37° C. The solution was transferred toEppendorf tubes and extracted with an equal volume of phenol 1×,phenol-CHCl₃ 1×, and CHCl₃ 1×, and ethanol precipitated. Pellets wereresuspended in 50 μl of TE buffer (10 mM Tris pH 8.0, 1 mM EDTA pH 8.0),and genomic DNA was analyzed by restriction digestion. One plaqueyielded the expected product.

This plaque of Av1ALH81 was plaque purified as follows. 5×10⁵ 293 cellsper well were plated on a 6-well tissue culture plate. The next day,medium was removed from the cells, and 0.4 ml of infection mediumcontaining 3 varying amounts of the resuspended plaque were added toeach well, 25 μl, 2.5 μl, and 0.25 μl. The plate was rocked for 1.5 hrsin a 37° C./5% CO₂ incubator, after which the media was removed, and thewells were overlaid with a 1:1 mixture of 2× MEM and 2% SeaPlaqueagarose as described. Plaques were visible in all wells 9 days afterinfection. Several plaques were picked from the lowest dilution well(0.25 μl of resuspended plaque), and screened for the presence ofAv1ALH81 as described. All plaques yielded the expected virus.

One plaque-purified plaque was selected for large scale viruspreparation. 5×10⁵ cells were plated in each well of a 6 well plate andthe next day infected with 50 μl of the resuspended plaque-purifiedplaque as described. Five days after infection, the CPE was complete,cells and medium were transferred to 15 ml conical tubes and subjectedto four freeze/thaw cycles, then cleared of cell debris bycentrifugation at 1000 rpm for 5 min. The resulting supernatant isreferred to as crude viral lysate #1 (CVL-1). This CVL was used toinfect a 150 mm plate containing approximately 2×10⁷ 293 cells asfollows.

Medium was replaced with 1.25 ml of Infection Medium plus 100 μl of CVL,and the plate was rocked for 1.5 hrs as described, after which 20 mls ofcomplete medium was added. Approximately 20 hrs after infection, the CPEwas complete, and cells and medium were transferred to a 50 ml conicaltube, spun for 5 min at 1000 rpm, supernatant was removed and saved, andthe cell pellet was resuspended in 5 ml of supernatant. After fourfreeze/thaw cycles, the CVL was removed of cell debris as described. Theresulting supernatant is referred to as CVL-2. 30-80% confluent 150 mmplates of 293 cells were infected using the CVL-2 as follows.

600 μl of CVL-2 was added to 38 mls of Infection Medium, medium wasremoved from the plates, and replaced with 1.25 ml of theCVL-2-Infection Medium mixture. Plates were rocked for 1.5 hrs asdescribed, after which 20 mls of complete medium was added to eachplate. The CPE was complete after 30 hrs and cells were processed asfollows. Cells and media were harvested into 250 ml centrifuge bottlesand spun at 1500 rpm for 10 min. The cell pellet was resuspended in 20mls of supernatant. Cells were lysed by five freeze/thaw cycles,followed by centrifugation in a SW40 rotor at 7000 rpm for 10 min at 4°C. Virus in the supernatant was purified on a CsCl step gradient asfollows.

3.0 ml of 1.25 g/ml CsCl in TD buffer (25 mM Tris, 137 mM NaCl, 5 mMKCl, 0.7 mM Na₂ HPO₄ Ph 7.5) was placed in four ultraclear Beckmann#344060 ultracentrifuge tubes. 3.0 ml of 1.4 g/ml CsCl in TD buffer wasthen underlaid. The CsCl layers were overlaid with 5.0 ml of viralsupernatant. Centrifugation was performed at 35,000 rpm, 22° C. for 1 hrin a SW40 rotor. Two bands were visible, an upper band consisting ofempty capsids, and a lower band composed of intact recombinantadenovirus.

The lower band was collected with a 3 ml syringe and a 18 gauge needle,and then rebanded by placing 8.0 ml of 1.33 g/ml CsCl in TD buffer intotwo ultracentrifuge tubes, and overlaying with virus collected from thefirst spin. Centrifugation was performed at 35,000 rpm, 22° C. for 18hrs. The viral band was collected as described and glycerol was added toa final concentration of 10%. The virus was dialyzed against one literof 10 Mm Tris pH 7.4, 10 Mm MgCl₂, and 10% glycerol at 4° C. Dialysislasted for four hours with buffer changes every hour. The virus wasrecovered and stored at -70° C. in aliquots in sterile Eppendorf tubes.The titer of this virus preparation (Lot # MS1-1) was 1.5×10¹¹ pfu/ml. Asecond Av1ALH81 viral prep was made in a similar manner as described,again using 600 μl of CVL-2 and 30-150 mm plates of 80% confluent 293cells. The titer of the second prep (Lot # MS1-2) was 9×10¹⁰ pfu/ml.

At this stage, the viral DNA is checked for deletions or rearrangements.Studies utilizing retroviral vectors containing Factor VIII cDNAsequences have been shown to delete and/or rearrange portions of theFactor VIII cDNA at high frequencies (Lynch et. al., 1993), and similarrearrangements may be seen with Factor VIII-containing adenoviralvectors. Therefore, viral DNA was isolated from both lots (MS1-1, andMS1-2) of AvALH81 as follows. 100 μl of purified virus was added to 100μl of TE, 5 μl of 10% SDS, and 20 μl of 10 mg/ml Proteinase K (Sigma),and digested overnight at 37° C. The viral DNA was extracted with anequal volume of phenol 1×, phenol-CHCl₃ 1×, and CHCl₃ 1×, then thesupernatant was put over a Centricon 10 concentrator (Amicon) and thevolume was increased to 2 mls with TE, and spun at 5000 rpm for onehour. The centricon was then washed with 2 mls of TE, and spun for 30min at 5000 rpm. DNA was recovered by inverting the upper chamber of thecentricon, inserting into the collection tube, and centrifugation at3000 rpm for 5 min. Final volume of the purified DNA was increased to100 μl, and the DNA concentration was calculated. 10 μg of MS1-1, MS1-2,and dl327 DNA was digested overnight with BamHI, HindIII, or, NdeI, andrun on a 0.8% agarose gel. DNA fragments were visualized with ethidiumbromide staining (FIG. 22). Both Av1ALH81 lots look the same, and allrestriction fragments are of the predicted sizes. Therefore, unlike theFactor VIII-containing retroviral vectors (Lynch et. al., 1993), thegenome of Av1ALH81 is stable.

The recombinant adenoviral vector Av1ALAPH81 was generated as outlinedin FIG. 23. 2×10⁶ 293 cells were cotransfected in a 100 mm tissueculture dish with 10 μg of the undigested shuttle plasmid, pAvALAPH81(FIG. 20). Generation of adenoviral vector Av1ALAPH81 then was carriedout in the same manner as the generation of adenoviral vector Av1ALH81.Av1APH81 can be generated in the same manner.

EXAMPLE 7 In Vivo Expression of Adenoviral Vectors Including DNAEncoding Factor VIII Plus Genomic Elements

A. Factor VIII Tri-Sandwich ELISA

Before Av1ALH81 could be tested for Factor VIII expression in vivo, inmice, or in vitro, in tissue culture cells, it was necessary to developan assay capable of measuring low levels of human Factor VIII present inmouse plasma. The only commercially available Factor VIII assay, Coatest(Kabi Pharmaceuticals) measures the biological activity of Factor VIIIprotein, and can be used to measure Factor VIII levels in tissue culturecells. However, Coatest cannot distinguish human Factor VIII from animalFactor VIII and, therefore, is not useful for measuring human FactorVIII in animal plasma. To measure the amount of human Factor VIIIpresent in tissue culture medium or animal plasma samples, aquantitative Factor VIII tri-sandwich ELISA was developed. This ELISAcan measure human Factor VIII specifically in mouse and dog plasma, andcan measure reproducibly Factor VIII concentrations down to 1.0 ng/ml.The assay is performed as follows.

A 96 well microtiter plate is coated with two commercially availablemonoclonal antibodies with unique epitopes for Factor VIII protein andincubated overnight at 4° C. to allow adherence to the plastic wells.0.5 ug of each antibody (N7, Biodesign; and ESH2, American Diagnostica)were diluted in dilution buffer (1.59 g Na₂ CO₃, 2.93 g NaHCO₃, sterileH₂ O to one liter, pH 9.6), and 100 μl of the dilution was added to eachwell. These antibodies constitute the primary antibody. The use of twocapture antibodies, that act cooperatively to increase the sensitivityof the assay, has not been described previously. After the overnightincubation, the plate is washed gently 3× with 200 μl per well of 1× PBSand blotted dry. Blocking agent 1× PBS, 10% Horse Serum (heatinactivated, BioWhittaker), and 1 mm CaCl₂ ! is added, and incubated fortwo hours at room temperature, after which the plate is washed with 200μl per well of washing solution 1× PBS, 0.05% Tween 20 (Sigma)! 3× andblotted dry. Samples then are diluted appropriately (usually a 5-folddilution) in TNTC (50 mm Tris pH 7.2, 5 mm CaCl₂, 0.1% Tween 20, 0.5 MNaCl), aliquoted into each well, and incubated for one hour at 37° C.,after which the wells are washed with the washing solution as described.The secondary antibody, which is diluted serum from a hemophiliac (a1:1000 dilution in the blocking agent solution, 100 μl per well)containing Factor VIII inhibitor antibodies, is added and allowed tobind for one hour at 37° C., after which the wells are washed with thewashing solution as described. The third antibody, a commerciallyavailable goat anti-human IgG antibody conjugated to horseradishperoxidase (goat anti-human IgG-HRP, Pierce, 0.8 mg/ml, diluted 1:5000in blocking agent, 100 μl per well), is added, and incubated for onehour at 37° C. The excess antibody then is washed out of the wells (asdescribed, but 5×) and the substrate tetramethylbenzidine (TMB)(Kirkegaard and Perry Labs; 100 μl of the commercially availablesolution), which when cleaved by the HRP, yields a blue color, is addedto each well. The level of color that develops is proportional to theamount of Factor VIII present in the sample. The reaction is stopped,after 2-3 minutes with an acid stop solution (TMB stop solution,Kirkegaard and Perry Labs, 100 μl per well) and the absorbance isdetermined using a microtiter plate reader. An example of a typicalstandard curve, using full-length human Factor VIII proteinconcentrations ranging from 0.078 ng/ml to 40.00 ng/ml is displayed inFIG. 24.

B. Half-Life Study of B-Domain Deleted Factor VIII in Mouse Plasma

After development of this extremely sensitive Factor VIII ELISA, ahalf-life study of B-domain deleted (BDD) Factor VIII in mouse plasmawas undertaken. It had been reported (Hoeben et. al., 1993) that thehalf-life of human Factor VIII in mice was only one hour, compared tothe 10 to 12 hour half-life of full-length human Factor VIII in humansand dogs (Brinkhous et al., PNAS, Vol. 82, pgs. 8752-8756 (1985)). Thedetermination of the half-life of BDD human Factor VIII in mice wasimportant for the subsequent evaluation of the efficacy of Av1ALH81 forgene therapy protocols utilizing the mouse as an in vivo model.

The half-life study was performed twice. In the first experiment (FIG.25) five C57bl/6 female mice were injected via tail vein with 400 ng ofBDD Factor VIII protein. Blood was drawn at 0.5, 1.5, 2.5. and 6.5 hourspost injection. In the second experiment (FIG. 26), designed to focus onthe 2 to 14 hour post injection time range, four C57bl/6 female micewere injected via tail vein, with 500 ng of BDD Factor VIII. Blood wasdrawn at 2, 5, 8, 12, and 14 hours post injection and plasma analyzedfor the presence of human Factor VIII antigen. The results are displayedin FIGS. 25 and 26. The half-life of human Factor VIII in mice wascalculated to be 4-5 hours. This result contrasts with the half-lifecalculated by Hoeben et. al. (1993). However, in the study by Hoeben et.al. (1993), the half-life of Factor VIII in mice was analyzed over onlya 2 hour time period. In the study reported here, it was found thatthere was a sharp decrease (half-life 1.7 hours) in the level of FactorVIII antigen in mouse plasma between 30 minutes and 2 hours postinjection (FIG. 25), with the decay leveling off to a half-life of 4-5hours at subsequent time points (FIGS. 25 and 26). Therefore, theresults indicate that the half-life of human BDD Factor VIII in mice isapproximately 2-3 times shorter than the human Factor VIII half-life inhumans and dogs.

C. Production of Biologically Active Factor VIII In Vitro

To determine if Av1ALH81 transduction resulted in the production ofbiologically active Factor VIII in vitro, 293 cells were infected withCVL-1, generated from two separate plaques of plaque-purified Av1ALH81as follows. The medium was removed from 3-150 mm plates of 293 cellscontaining 1.5×10⁷ cells, and replaced with 1.15 mls of InfectionMedium, plus 100 μl of CVL-1 from either Av1ALH81 plaque (plaque 1 orplaque 2), or, for the negative control plate, 1.25 mls of InfectionMedium. Plates were rocked for 1.5 hrs. as described, after which 20 mlsof complete medium was added to each plate. 1.0 ml of medium wascollected from each plate at 0, 12, and 24 hr. time points, and analyzedfor the presence of Factor VIII antigen, using the human FactorVIII-specific ELISA, described above, and analyzed for biologicalactivity, using the Coatest Assay (Kabi Pharmaceuticals). The resultsare displayed in Table II below.

                  TABLE II    ______________________________________    Expression of Factor VIII in Av1ALH81 Transduced 293 Cells                  Assay                        ELISA (ng/ml)                                   Coatest (ng/ml)*    Virus   Time (hrs)  total antigen                                   biological activity    ______________________________________    AV1ALH81             0          0.0        0.0    plaque 1            12          9.8        6.9            24          10.2       1.0    AV1ALH81             0          0.0        0.0    plaque 2            12          22.1       7.6            24          24.3       0.0    No virus             0          0.0        0.0            12          0.0        0.0            24          0.0        0.0    ______________________________________     *converted from units in which one unit of activity equals 200 ng/ml of     Factor VIII.

As shown in Table II, the cells produced 10-20 ng/ml of Factor VIIItotal antigen as determined by ELISA, and at 12 hrs., 7 ng/ml of FactorVIII was biologically active. However, by 24 hours, the biologicalactivity was lost. The lower level of biologically active Factor VIII at12 hours and the lack of active Factor VIII at 24 hours can be explainedby the fact that the cells were undergoing a cytopathic effect thatstarted at 12 hours and was complete by 24 hours. Therefore, de novosynthesis of Factor VIII had probably begun to decrease at 12 hours andthe Factor VIII present in the medium was becoming degraded by 24 hours.

D. In Vivo Expression of BDD Factor VIII From Av1ALH81

To determine if human BDD Factor VIII was expressed from Av1ALH81 invivo, and if so, to follow the time course of Factor VIII expression, 15C57bl/6 female mice were injected with Av1ALH81. The virus was dilutedin injection medium (IMEM+1% FBS) to a total volume of 0.5 ml. Five micereceived a dose of 1×10¹⁰ pfu (67 μl of virus; concentration of 1.5×10¹¹pfu/ml), five mice received a dose of 4×10⁹ pfu (27 μl virus) and fivemice received 1×10⁹ pfu (7 μl virus). The viral suspension was infusedvia tail vein over a ten second period using a 0.5 ml syringe and a 27gauge needle. The control mouse received no injection. One mouse thatreceived 1×10¹⁰ pfu of Av1ALH81 died two days after injection. Blood wastaken from each mouse at one week intervals and analyzed for thepresence of human Factor VIII antigen by ELISA. The results of theanalysis, for the first five weeks post injection, is displayed in TableIII below, and graphically in FIG. 27.

                  TABLE III    ______________________________________    In Vivo Expression of Factor VIII              ELISA (Factor VIII ng/ml)    Mouse Virus Dose                    Week 1  Week 2                                  Week 3                                        Week 4                                              Week 5    ______________________________________     1    .sup. 1 × 10.sup.10                    53.3    19.3  0.0   4.3   0.0     2    .sup. 1 × 10.sup.10                    45.9    54.6  0.0   34.3  0.0     3    .sup. 1 × 10.sup.10                    34.5    35.3  3.0   2.6   0.0     4    .sup. 1 × 10.sup.10                    33.1    31.1  10.9  7.7   1.8    Mean  --        41.7    35.1  3.5   12.2  0.5     5    4 × 10.sup.9                    18.9    7.3   0.0   14.9  1.1     6    4 × 10.sup.9                    13.0    6.2   5.1   7.9   3.1     7    4 × 10.sup.9                    9.8     5.2   3.1   12.4  5.1     8    4 × 10.sup.9                    25.9    13.1  3.4   18.4  12.9     9    4 × 10.sup.9                    17.1    5.9   0.5   9.7   6.0    Mean  --        16.9    7.5   2.3   12.6  5.6    10    1 × 10.sup.9                    0.8     0.0   0.0   3.6   0.8    11    1 × 10.sup.9                    0.4     0.0   0.5   5.1   0.3    12    1 × 10.sup.9                    1.1     1.6   2.2   4.2   2.5    13    1 × 10.sup.9                    1.0     1.7   0.0   1.7   1.3    14    1 × 10.sup.9                    1.4     0.9   0.0   1.9   0.0    Mean  --        0.9     0.9   0.5   3.3   1.0    Control          0         0.0     0.0   0.0   0.0   0.0    ______________________________________

The mice receiving the highest viral dose (1×10¹⁰ pfu) were producing 42and 35 ng/ml human BDD Factor VIII one and two weeks post injection,respectively. If these values are corrected for the difference inhalf-life of human Factor VIII in mice (4-5 hrs., see above) compared tohumans (10-12 hrs.), levels in the plasma are adjusted to 126 and 96ng/ml of Factor VIII at one and two weeks, respectively. Physiologicallevels of Factor VIII in humans is ˜100-200 ng/ml and therapeutic levelsare ˜10 ng/ml. Therefore, these mice are producing physiological levelsof Factor VIII. In addition, the mice that received the lower dose of4×10⁹ pfu of Av1ALH81 are producing Factor VIII protein well overtherapeutic levels. The expression of human Factor VIII, in an animalmodel, has never before been demonstrated.

Three to five weeks post injection, however, the Factor VIII levels haddecreased significantly. To determine if this decrease in Factor VIIIexpression at three weeks was due to an immunological response to theFactor VIII antigen, a second half-life study was performed usingfull-length human Factor VIII protein. The four mice that had receivedthe highest viral dose (1×10¹⁰ pfu), and four control mice wereinjected, via tail vein, with 500 ng of full-length human Factor VIII.Blood was drawn at 1, 2, 4, 8, and 12 hours post injection and analyzedfor the presence of human Factor VIII antigen by ELISA. FIGS. 28 and 29display the results of this analysis. The half-life of full-lengthFactor VIII is similar in both sets of mice, and can be calculated to beabout 3.0 hrs. Two conclusions can be drawn from these data: 1) Thehalf-life of full-length Factor VIII and BDD Factor VIII in mouse plasmaare comparable, 3 hrs. and 4-5 hrs., respectively, and 2) the loss inFactor VIII expression in the mice at 3 weeks is not due to rapidclearance of the Factor VIII by specific antibodies in mouse plasma,and, therefore, may be due to the loss of vector from the liver (or lossof the cells containing the vector), or reduced transcription of theFactor VIII cDNA.

The time course of Factor VIII expression in mice was repeated, and thedata are shown in FIG. 30.

EXAMPLE 8

Fifteen C57b1/6 female mice were injected via tail vein 4×10⁹ pfu (27 μlof virus) of Av1ALH81 in injection medium (IMEM+1% FBS). The viralsuspension was infused via the tail vein over a ten second period usinga 0.5 ml syringe and a 27 gauge needle. A control mouse received noinjection. Blood was taken from each mouse at one week intervals andanalyzed for the presence of human Factor VIII antigen by ELISA. Theresults are shown graphically in FIG. 30.

EXAMPLE 9 Construction of Adenoviral Vectors Including Factor IXSequences with Genomic Elements

Vectors were prepared in which the Factor IX sequences incorporatedgenomic elements, i.e., sequences from the human Factor IX gene. Theseelements included the 5' untranslated region, a centrally truncatedfirst intron, the full 3' untranslated region and naturally-occurringpolyadenylation site. The 5' genomic elements were obtained by PCRamplication using genomic Factor IX clones as templates. The three primeuntranslated region was obtained from a plasmid provided by Dr. Hsueh(Shanghai, China). An alternative approach, which can be used to readilyobtain these elements, is to PCR amplify them from human genomic DNA.

A Factor IX sequence, which includes 9 bp of the Factor IX promoter, the5' untranslated region, the coding region, and a 162 bp segment of the3' untranslated region, was excised from pKG5IX-2 (obtained from GeorgeBrownlee, University of Oxford, Oxford England) as a Bam HI to HindIIIfragment. This fragment is described further in Anson et al., Nature,Vol. 315, pgs. 683-685 (1985). This insert was inserted into thepolylinker of pBluescript II SK+ (Stratagene) to form BLSKH9CI. (FIG.31). The Factor IX sequences were sequenced completely and verified tobe correct. Factor IX DNA with genomic elements could also have beenobtained according to the procedures disclosed in U.S. Pat. No.4,994,371 and European Patent EP 0 107 278 B1.

A fragment containing the downstream part of the coding sequence, thefull 3' untranslated region, the native Factor IX polyadenylationsignal, and 331 bp past the polyadenylation site was excised frompCMVIXa (provided by Jerry Hsueh, Fudan University, Shanghai, China)with PpuMI and BglII. The BglII single strand overhang was blunted.pBLSKH9CI was cut with PpuMI and HindIII, the HindIII site was blunted,and the backbone fragment was joined to the fragment obtained frompCMVIXa as a PpuMI-blunt ligation. The resulting plasmid, pBLSKH9D (FIG.32), contains the 9 bp of promoter, 5' untranslated region, the entireFactor IX coding sequence, the full 3' untranslated region, naturalpolyadenylation signal, and 331 bp downstream from the polyadenylationsignal.

To generate constructs that contain a centrally truncated first intron,a cloning intermediate was prepared. This intermediate removed a Bc1Isite downstream from the coding sequence to enable cloning into anupstream Bc1I site. The 3' end of the cDNA in pB1SKH9CI was removed fromPpu MI to HindIII. The single strand overhangs in the plasmid backbonewere blunted and ligated to yield pBLH9CINT. (FIG. 33)

The 5' end of the cDNA in pBLH9CINT was modified to contain thecentrally truncated first intron with a 3 way ligation using 2 PCRgenerated fragments. These fragments were generated using phage preps astemplates (Yoshitake, S et al., 1985, Biochemistry: 24, 3736-3750). Thetwo PCR generated fragments contained (5' to 3'):

1) SpeI site, SalI site, full 5' untranslated region, first exon of theFactor IX gene, the first 991 bp of the Factor IX first intron, and anAatII site.

2) AatII site, the last 448 bp of the Factor IX first intron, and partof the Factor IX second exon extending past the naturally occurring Bc1Isite in the upstream part of this exon.

PCR fragment 1 was digested with SpeI and AatII. PCR fragment 2 wasdigested with AatII and Bc1I. BLH9CINT was digested with SpeI and Bc1Iand the backbone fragment was isolated. The three fragments were joinedwith a 3 way ligation to yield the plasmid pBLH9EINT. (FIG. 34) Thisplasmid contains the 5' untranslated region of Factor IX, the firstexon, the centrally truncated first intron, and the coding sequence upto the PpuMI site.

To generate pBLH9E (FIG. 35), the 3' end of the coding sequence wasre-inserted. The 3' end of the Factor IX sequence was excised frompBLSKH9CI and inserted into the pBLH9EINT backbone as an AvaI-AvaIfragment. The resulting plasmid pBLH9E (FIG. 35) contained the Factor IX5' untranslated region, first exon, centrally truncated first intron,remainder of the coding sequence, and 162 bp of 3' untranslated region.

To generate pBLH9F (FIG. 36), a fragment containing the 3' end of thecoding sequence and the full 3' untranslated region was excised frompBLSKH9D and inserted into the pBLH9EINT backbone as an AvaI-AvaIfragment. Thus pBLH9F has the 5' untranslated region, first exon,truncated first intron, remainder of the coding sequence, full 3'untranslated region, and 300 bp downstream from the polyadenylationsite.

The Factor IX sequences were then excised from pBLSKH9D, pBLH9E, andpBLH9F and inserted into the pAvS6 backbone as SpeI-ClaI fragments. Theresulting plasmids were termed pAV1H9D (FIG. 37), pAV1H9E, and pAV1H9F,respectively. However, when pAv1H9E and pAv1H9F were sequenced, errorswere found in the 5' untranslated region of the Factor IX gene. Theseerrors were repaired. The sequence errors were traced back to pBLH9EINT.Miniprep one this plasmid had been used to generate the subsequentplasmids. pBLH9EINT miniprep six was found to have the correct sequence.The SpeI to AatII fragment in pBLH9EINT miniprep six was used to replacethe corresponding fragment in pAv1H9E and pAv1H9F to yield pAv1H9ER(FIG. 38) and pAv1H9FR (FIG. 39), respectively. These plasmids containthe adenovirus type 5 ITR, RSV promoter, tripartite leader, Factor IXsequence, SV40 polyadenylation site (which is superfluous in pAV1H9D andpAV1H9FR), and adenovirus homologous recombination region.

pAV1H9D, pAV1H9ER, and pAV1H9FR were then used to generate adenoviralvectors by procedures hereinabove described. Briefly, linearizedplasmids were co-transfected with the large ClaI-cut fragment of Addl327 into 293 cells. Plaques were selected, expanded, and screened. Thethree vector isolates chosen were termed Av1H9D, Av1H9ER, Av1H9FR. Thesewere grown into large scale preps and plaque titered on 293 cells.

EXAMPLE 10

2×10⁸ pfu of Av1H9B, Av1H9D, Av1H9ER, or Av1H9FR were injected via tailvein into C57B1/6 mice. Two mice received each virus. One week later,plasma was sampled by ELISA and an immunochromogenic bioassay describedabove for human Factor IX antigen and biological activity, respectively.The ELISA results, shown in FIG. 40, demonstrate that the inclusion ofgenomic elements dramatically increased Factor IX expression. TheAv1H9FR vector effected the most expression which was more than 200 foldgreater than the level obtained with Av1H9B. The immunochromogenic assayresults for eight experimental mice and one negative control mouse(which was injected with the beta-galactosidase vector, Av1LacZ4) areshown in Table IV below.

                  TABLE IV    ______________________________________                   Plasma Factor IX (ng/ml)    Vector Mouse Number  ELISA   Immunochromogenic    ______________________________________    Av1H9B 1             <8      <8    Av1H9B 2             <8      <8    Av1H9D 3             74      65    Av1H9D 4             48      49    Av1H9ER           5             116     102    Av1H9ER           6             101     80    Av1H9FR           7             1,339   1,051    Av1H9FR           8             1,467   1,391    Av1LacZ4           9             <8      <8    ______________________________________

These results demonstrate that the human Factor IX expressed fromAv1H9D, AV1H9ER, and AV1H9FR was functional. Livers were collected oneweek after vector injection and DNA and RNA were prepared. Southernanalysis demonstrated an average of 1-2 vector copies per liver cell forall four vectors. (Data not shown.) Northern analysis revealed RNAspecies of the correct size for each vector with band intensities thatparallelled the Factor IX plasma levels. (Data not shown.)

EXAMPLE 11 Persistent High Level Expression of Human Factor IX in MiceWith Vectors With Genomic Elements

1×10⁹ pfu of Av1H9B, Av1H9D, or Av1H9ER were injected via tail vein intoC57B1/6 mice. 1×10⁹ pfu of Av1H9FR were also administered to C57B1/6mice via tail vein injection. The cohort size for each regimen was 5mice. At the indicated time points plasma was obtained and assayed forhuman Factor IX by ELISA.

The results, shown in FIGS. 41 and 42, again demonstrate that theinclusion of genomic elements in the Factor IX sequences dramaticallyincreased Factor IX expression. Factor IX levels that approach normalwere obtained with the low dose of 1×10⁹ pfu of Av1H9D and Av1H9ER, andAv1H9FR yielded very high supraphysiologic levels. In each case,high-level expression at or above the therapeutic level from Av1H9D,Av1H9ER, and Av1H9FR persisted for the 8 to 10 month duration of theexperiment. This exceeded the 7 week duration of expression which hadpreviously been achieved with higher doses of Av1H9B.

EXAMPLE 12

Expression from Av1H9B, Av1H9D, and Av1H9ER was tested in tissueculture. HepG2 and HeLa cells were transduced at an moi of 2 (2 pfu percell). 48 hours later the medium was collected and assayed by ELISA forhuman Factor IX. The data, shown in FIG. 43, demonstrate that theincremental improvements seen in HepG2 cells with Av1H9D and Av1H9ERcorrespond to those seen in vivo in mice that received 2×10⁸ pfu ofvirus. In HeLa cells, the inclusion of the 3' untranslated region(Av1H9D) had no effect, whereas inclusion of the intron (Av1H9ER)improved expression dramatically.

EXAMPLE 13 High Level Tissue-Specific Expression of Functional HumanFactor VIII in Mice

Cell Culture, Viral Transduction, and Metabolic Labeling

293 cells were cultured in IMEM (Improved Minimal Essential Medium) plus10% FBS. HepG2 cells were cultured in EMEM (Eagle Minimal EssentialMedium), 10% FBS, 1 mM nonessential amino acids, and 10 mM Na Pyruvate.For viral transduction, 1×10⁷ HepG2 cells, plated on 100 mm dishes, weretransduced at a multiplicity of infection (MOI) of 30. Medium wasremoved from the cells, and 0.4 ml of Infection Medium (IMEM plus 1%FBS) plus the indicated vector, or without vector for mock infections,was added. The plates were incubated with rocking for 1.5 hours, afterwhich 10 ml of HepG2 culture media were added. 12 hours later, mediumwas changed, and 24 hours later, medium was assayed for Factor VIII(FVIII) activity, and cells were collected for RNA analysis. Metaboliclabeling of the HepG2 cells was performed 12 hours after transduction,using ³⁵ S-methionine as described (Pittman et al., Methods inEnzymology, Vol. 222, pg. 236 (1993)). The conditioned medium was thencollected, and the labeled BDD FVIII was purified by immunoprecipitation(Pittman and Kaufman, 1993). Where indicated, the immunoprecipitateswere treated with 7.5 U/ml of thrombin (Haematologic Technologies, EssexJct., Vt.) for one hour at 37° C. (Pittmanet al., 1993).

Animal Procedures

All experiments involving mice adhered to protocols approved by theInstitutional Animal Care and Use Committee in accordance with theAnimal Welfare Act. C57BL/6 female mice, 3-6 weeks old, were purchasedfrom Harlan Sprague Dawley. Tail vein injections were performed with theindicated doses of vector in 0.5 ml of Infection Medium. At the timepoints indicated, blood was obtained from the retroorbital plexus.Sodium citrate was immediately added to a final concentration of 0.38%(w/v) and the samples were placed on ice for no longer than 30 minutes.The samples were centrifuged for five minutes in an Eppendorf Microfugeafter which the plasma was collected, aliquoted, and frozen.

Assays for Human FVIII

Total human FVIII antigen was quantitated by ELISA as described(Connelly et al., Human Gene Therapy, Vol. 6, pgs. 185-193 (1995)).Full-length recombinant FVIII protein generously supplied by GeneticsInstitute (Cambridge, Mass.) was used to generate a standard curveranging from 1 to 100 ng/ml. BDD FVIII protein (supplied by GeneticsInstitute, Cambridge, Mass.) and full-length recombinant FVIII weresimilarly quantified by this ELISA. Normal mouse plasma did notinterfere with the assay and the limit of sensitivity with mouse plasmasamples containing BDD FVIII was 3 to 6 ng/ml. Mouse plasma samples werenormally diluted 1:5 or 1:10 for the ELISA.

Biologically active human FVIII was measured using the Coatestchromogenic bio-assay (Chromogenix, Molndal, Sweden) as directed.Coatest measures the FVIII-dependent generation of Factor Xa from FactorX, with one unit defined as the amount of FVIII activity in one ml ofpooled human plasma, 100 to 200 ng/ml (Vehar et al., Biotechnology ofPlasma Proteins, Albertini et al., eds. pg. 2155, Basel, Karger, 1991).Pooled human plasma (George King Bio-Medical, Inc., Overland Park,Kans.) was used as the FVIII activity standard.

Southern Blot and RNAse Protection Analyses

DNA was isolated from mouse livers using standard procedures. Briefly,livers were minced and incubated overnight in SDS/Proteinase K buffer.This was followed by three phenol/chloroform extractions, one chloroformextraction, ethanol precipitation and resuspension in water. 10 μg ofeach DNA sample were digested with Bam HI and subjected to Southernanalysis. The probe, prepared by random oligonucleotide priming,contained human FVIII cDNA sequences from +73 to +1345 (Toole et al.,1984; Wood et al., 1984). The copy number control standards wereprepared by adding 3.0 ng, 1.5 ng, 600 pg, and 60 pg of viral DNA,equivalent to 50, 25, 10, and 1 vector copies per cell, respectively to10 μg of uninjected control mouse liver genomic DNA and digesting withBam HI. The band intensities were quantitated with a Molecular DynamicsPhosphorImager SF.

RNA was isolated from mouse livers using the RNAzole B (TelTest,Friendswood, Tex.) extraction method. RNAse protection analyses wereperformed using the RNAse Protection Kit II (Ambion, Austin, Tex.). Foreach sample, quantities of 5 to 150 μg of total cellular RNA washybridized for 12 hrs at 45° C. with 5×10⁴ cpm of a gel-purified RNAprobe (see below), digested with the RNAse A/T1 solution provided withthe kit, diluted 1:100, processed as directed, and analyzed on an 8%polyacrylamide-8M urea gel (SequaGel, National Diagnostics, Atlanta,Ga.). The band intensities were quantitated with a Molecular DynamicsPhosphorImager SF. The values obtained were normalized for the number ofG-residues in the protected mRNA fragment, as the antisense RNA probeswere synthesized with α-³² P-CTP. RNA molecular weight markers weresynthesized using the RNA Century Marker Template Set (Ambion, Austin,Tex.). ³² P-labeled fragments from Hpa II digested pBR322 were used asDNA size markers. The FVIII probe template (FIGS. 46 and 48), pGemSRpr,was constructed by inserting the 204 bp Sac I-Eco RI fragment isolatedfrom pMT2LA (provided by Genetics Institute, Cambridge, Mass.) (Toole etal., Prot. Nat. Acad. Sci., Vol. 83, pg. 5939 (1986) into pGem4Z(Promega, Madison, Wis.) cut with Sac I and Eco RI. The ALAPH81 probetemplate (FIG. 47), pGEMALAPF8pr, was created by digesting pAVALAPH81(FIG. 20) with Mse I (filled in with T4 DNA polymerase) and Eco RI. This506 bp fragment containing part of the albumin promoter, the ApoA1 firstexon, first intron, second exon, and FVIII coding region sequences wasinserted into pGem4Z (Promega, Madison, Wis.) digested with Sma I andEco RI. The ALH81 probe template (FIG. 47), pGemF8probe, has beendescribed (Connelly et al., 1995). The mouse GAPDH-specific and mouseactin-specific probe templates were generated from the pTRI-GAPDH mouseplasmid (Ambion, Austin, Tex.) and pTRI-ACTIN mouse plasmid (Ambion,Austin, Tex.) digested with Sty I and Hind III, respectively. TheFVIII-specific probe templates were linearized with Hind III and allanti-sense RNA probes were synthesized with SP6 polymerase and α-³²P-CTP (Amersham, Arlington Heights, Ill., 3000 Ci/mmole).

Characterization of Human FVIII Expressed in Vitro

The expression of human BDD FVIII in HepG2, human hepatoma cells,transduced with either Av1ALH81 (FIG. 21) or Av1ALAPH81 (FIG. 23) wascompared (Table V). The biological activity of the BDD FVIII present inthe conditioned medium was analyzed using the Coatest chromogenicbioassay (Chromogenix, Molndal, Sweden).

                  TABLE V    ______________________________________    Vector     Biological Activity (mU/10.sup.6 cells/24 hrs)    ______________________________________    Av1ALAPH81 2375               2167    Av1ALH81   1716               1694    Av1ALH9B     0    Mock         0    ______________________________________

As shown in Table V, in duplicate infections with Av1ALH81 andAv1ALAPH81, >1600 or >2100 mU per 10⁶ cells per 24 hours, respectively,were detected, revealing that transduction with both vectors resulted inthe production of high levels of biologically active FVIII. No FVIII wasdetected in mock infected cells or cells transduced with a controlvector, Av1ALH9B (Connelly et al., 1995), a recombinant adenovirusencoding the human factor IX (FIX) cDNA (Table V).

The structure of the FVIII protein produced by HepG2 cells was studiedby metabolic labeling of the cells after transduction with eachFVIII-encoding vector, and subsequent immunoaffinity purification of thelabeled BDD FVIII protein from the conditioned medium (FIG. 44). Thecells were exposed to ³⁵ S-methionine and the labeled BDD FVIII waspurified by immunoprecipitation (Pittman, et al., 1993). Theimmunoprecipitates before (-) or after (+) treatment with thrombin(Pittman and Kaufman, 1993) were analyzed by reducing SDS polyacrylamidegel electrophoresis (lanes 1-6). For comparison, purified BDD FVIIIprotein (5.5 μg/lane; a gift from Genetics Institute, Cambridge, Mass.),before (-) or after (+) treatment with thrombin was analyzed on the samegel as the immunoprecipitates. After electrophoresis, the gel wasCoomassie stained and divided. The portion containing the purifiedprotein and protein size markers was dried and photographed (lanes 7 and8), while the portion containing the metabolically labeledimmunoprecipitates was analyzed by autoradiography (lanes 1-6). Thearrows labeled SC, HC, and LC indicate the BDD FVIII single-chain,heavy-chain, and light-chain polypeptides (Pittman et al., 1993).Protein molecular weight standard (lane 9; Bio-Rad, Hercules, Calif.)sizes are indicated in kilodaltons (Kd).

The BDD FVIII protein secreted by both Av1ALH81- andAv1ALAPH81-transduced cells was composed of three prominent species,representing the 170 Kd single chain, the 92 Kd heavy chain, and the 80Kd light chain (FIG. 44, lanes 1 and 3) and was directly comparable tothe polypeptide pattern observed with the purified BDD FVIII proteinisolated from stably transfected CHO cells (FIG. 44, lane 7; Pittman etal., 1993). Digestion with thrombin resulted in the disappearance of thesingle chain, and the generation of 73, 50, and 43 Kd polypeptides (FIG.44, lanes 2 and 4), which was identical to the thrombin cleavage patternof the purified BDD FVIII protein (FIG. 44, lane 8; Pittman et al.,1993). No FVIII-specific peptides were detected in Av1ALH9B-transducedor mock infected cells (FIG. 44, lanes 5 and 6). The >200 Kd polypeptide(FIG. 44, lanes 1-6) probably represents non-specific copurification offibronectin (Connelly et al., 1995) and was not detected in the purifiedprotein samples (FIG. 44, lanes 7 and 8). RNAse protection analysisverified that transcription initiation of the BDD FVIII mRNA produced inAv1ALH81-transduced HepG2 cells occurred at the predicted site and thatthe Av1ALAPH81-derived mRNA was accurately spliced (data not shown).Therefore, in vitro transduction of cells with both FVIII-encodingvectors resulted in the production of high levels of authentic,biologically active human BDD FVIII.

In Vivo Expression of Human FVIII

To compare the expression of human BDD FVIII in mice injected with theFVIII-encoding adenoviral vectors, a short-term study was designed inwhich 4×10⁹ pfu of Av1ALH81 or Av1ALAPH81 was administeredintravenously, via tail vein, to groups of eight mice each. Since theCoatest assay does not distinguish human FVIII activity from mouseFVIII, an ELISA specific for full-length and BDD FVIII was developed(Connelly et al., 1995) and used to measure human FVIII in mouse plasma.The results of such assay are shown in FIG. 45. Data are plotted as amean value and standard deviation at each time point. Four mice for eachgroup were sacrificed one week after vector injection for subsequentDNA/RNA analysis. One week post injection, human FVIII levels in theplasma of mice injected with Av1ALH81 averaged 307±93 ng/ml (FIG. 45).However, plasma FVIII levels in mice that had received Av1ALAPH81averaged 1046±163 ng/ml (FIG. 45). Normal FVIII levels in humans are 100to 200 ng/ml (Vehar et al., 1991), and therapeutic levels are as low as10 ng/ml (Vehar et al., 1991). Therefore, mice that received Av1ALAPH81were producing human FVIII at levels one hundred fold higher than humantherapeutic levels, and three times higher than mice injected with anidentical dose of Av1ALH81. No human FVIII was detected in uninjectedcontrol mice (data not shown) or three mice injected with 4×10⁹ pfu ofAv1ALH9B (FIG. 45). FVIII levels in the mouse plasma were analyzedweekly for four weeks at which time the experiment was terminated. Atthree to four weeks post injection, FVIII levels in both sets of micehad decreased 30% from the peak expression level detected at one week(FIG. 45).

To verify that the liver transduction efficiency of the two FVIIIvectors was the same, two groups of four mice each injected with eithervector were sacrificed one week post injection and liver DNA was assayedfor the presence of the vector by Southern analysis (FIG. 46A). Each DNAsample was digested with BamHI prior to Southern analysis. The arrowdesignates a 3.2 kb fragment containing Av1ALH81-derived Factor VIIIsequences. Av1ALAPH81-derived sequences generated a 3.4 kb fragment.Lanes 5 and 6 represent liver DNA from an uninected control mouse, and amouse that received the control vector, Av1ALH9B, respectively. Lanes1-4 contain digested Av1ALH81 viral DNA in amounts equivalent to 50, 25,10, and 1 vector copies per cell. DNA markers (Gibco-BRL, Gaithersburg,Md.) sizes are indicated in kilobases (kb).

Comparison to vector copy number standards (FIG. 46A, lanes 1-4) showedan average of 18 vector copies per cell for both Av1ALH81- (FIG. 46A,lanes 7-10) and Av1ALAPH81- (FIG. 46A, lanes 11-14) transduced liver DNAsamples, revealing that both vectors transduced the mouse livers to asimilar level. No FVIII-containing vector was detected in an uninjectedcontrol mouse liver DNA sample (FIG. 46A, lane 5), or a liver DNA sampleisolated from a mouse injected with Av1ALH9B (FIG. 46A, lane 6).

The relative levels of human FVIII mRNA produced in the mice wasdetermined by RNAse protection analysis using RNA isolated from themouse livers (FIG. 46B), and an anti-sense RNA probe encoding aninternal portion of the FVIII coding region shared between both vectors(FIG. 46C). 30 μg of total cellular RNA isolated from the mouse liverswere used in each reaction. The arrow labeled FVIII designates the 212nt human FVIII-specific protected probe fragment. Lanes 1 and 12 contain³² P-labeled DNA and RNA molecular weight markers. Lane 2 containsundigested full-length probe. Lane 3 contains liver RNA from anAv1ALH9B-injected control mouse. Exposure shown is 5 days withintensifying screens. The lower panel displays a separate RNAseprotection assay, using 30 μg of total cellular mouse liver RNA, and ananti-sense RNA probe encoding a portion of the mouse glyceraldehyde3-phosphodehydrogenase (GAPDH) cDNA. The arrow labeled GAPDH designatesthe 134 nt mouse GAPDH-specific protected probe fragment. Exposure shownis 6 hours with intensifying screen.

With this probe, human FVIII-specific mRNA should protect a 212 ntfragment. A fragment of this size was seen in each of the FVIII-encodingvector-transduced mouse liver RNA samples (FIG. 46B, lanes 4-11), andnot in the Av1ALH9B-injected control mouse liver sample (FIG. 46B, lane3). Quantitation of the FVIII-specific mRNA protected probe fragments byphosphoimager scanning showed an average of 15-fold more RNA in theAv1ALAPH81-derived samples (FIG. 46B, lanes 8-11), compared to theAv1ALH81-transduced RNA samples (FIG. 46B, lanes 4-7). As an internalRNA standard, the mouse liver RNA samples were analyzed in a separateRNAse protection assay using an anti-sense RNA probe containing aportion of the mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH)coding region to verify the integrity of the RNA in each sample (FIG.46B, lower panel). Therefore, the addition of an ApoA1 intron to the BDDFVIII cDNA resulted in 3-fold higher plasma FVIII levels, and a 15 foldincrease in FVIII-specific mRNA in the livers of mice injected withAv1ALAPH81, compared to the FVIII protein and mRNA levels detected inmice that received an identical dose of Av1ALH81.

To verify that transcription from the albumin promoter initiated at thepredicted site in vivo (Gorski et al., Cell, Vol. 47, pg. 767, 1986;Connelly et al., 1995), an RNAse protection analysis was performed usingan anti-sense RNA probe containing sequences from the albumin promoterand the 5' end of the BDD FVIII cDNA (FIG. 47B; Connelly et al., 1995).

FIG. 47A depicts the RNase protection analysis results. RNA samplesisolated from mice that received Av1ALH81 (Lanes 4-7) were analyzedusing the ALH81 probe. The arrow labeled initiation designates the 247nt protected probe fragment indicating transcripts properly initiated atthe albumin promoter. RNA isolated from Av1ALAPH81-injected mice (Lanes11-14) was analyzed using the ALAPH81 probe. The 471 nt and 239 ntprotected probe fragments, representing unspliced and splicedtranscripts, respectively, are indicated. Liver RNA purified from amouse injected with the control vector, Av1ALH9B, analyzed with ALH81(Lane 3) and ALAPH81 (Lane 10) probes served as the negative control.Lane 1 contains ³² P-labeled RNA molecular weight markers. Lanes 8 and15 contain ³² P-labeled DNA molecular weight markers. Lanes 3 and 9contain undigested, full-length ALH81 and ALAPH81 probes, respectively.FIG. 47B is a schematic diagram depicting the probe templates andcomplementary mRNA fragments. The boxes marked Alb and SP6 represent thealbumin and SP6 promoter regions, respectively. The rightward pointingarrow indicates the site of transcription initiation from the albuminpromoter. The leftward pointing arrow indicates the direction oftranscription from the SP6 promoter. The solid black boxes represent theApo A1 genomic sequences.

Analysis of liver RNA samples isolated from mice injected with Av1ALH81revealed a fragment of the predicted size for transcripts properlyinitiated at the albumin promoter (247 nts; FIG. 47B, lanes 4-7;Connelly et al., 1995), and was not found in RNA isolated fromAv1ALH9B-transduced mouse liver (FIG. 47A, lane 3). The accuracy andefficiency of splicing of Av1ALAPH81-derived transcripts were evaluatedusing an anti-sense RNA probe capable of distinguishing unspliced FVIIImRNA from spliced mRNA (FIG. 47B) in an RNAse protection analysis (FIG.47A). With this probe, unspliced RNA would protect a fragment of 471nts. Transcripts accurately spliced would produce two protectedfragments, one 34 nts, representing the ApoA5' exon, and a fragment of239 nts derived from the 3' exon and FVIII coding region. AllAv1ALAPH81-derived RNA samples contained protected fragmentsrepresenting unspliced and spliced transcripts in approximately equalamounts (FIG. 47A, lanes 11-14). Quantitation by phosphorimagerscanning, and adjustment of the values for the number of G residues ineach protected fragment showed that an average of 69% of the transcriptswere spliced. Therefore, transcription from the albumin promoterinitiated at the predicted site in vivo, and accurate splicing of theApo A1 intron sequences from the FVIII pre-mRNA occurred in the mouselivers.

Liver-specific Expression of the Albumin Promoter

The modified mouse albumin promoter (Hafenrichter et al., Blood, Vol.10, pg. 3394 (1994); Connelly et al., 1995) had been shown to direct ahigh level of liver-specific expression in a conditionally transformedhepatocyte cell line, H2.35 (Zaret et al., 1998), under differentiatingconditions (DiPersio et al., 1991), and was active when transferred torat hepatocytes in vivo, in the context of a retroviral vector(Hafenrichter et al., 1994). However, the function of the mouse albuminpromoter in vivo, when incorporated into an adenoviral vector backbonehad not been determined. To ascertain whether expression from thealbumin promoter was tissue-specific, an RNAse protection analysis wasperformed using RNA isolated from several Av1ALAPH81-transduced mouseorgans. It had been shown previously, however, that intravenousinjection of adenoviral vectors to mice resulted in preferentialaccumulation of vector in the liver with other organs having lowertransduction efficiencies (Smith et al., Nature Genetics, Vol. 5, pg.397 (1993)). Therefore, first it was necessary to determine the relativetransduction efficiencies of the different organs, and then normalizethe RNA concentrations for the RNAse protection assay dependent upon theorgan transduction efficiency. DNA was isolated from liver, lung, andspleen derived from the Av1ALAPH81-injected mice one week postinjection, and the vector copy number per cell was assessed by Southernanalysis (FIG. 48A).

Each DNA sample was digested with Bam HI and subjected to Southernanalysis. Li, Lu, and Sp indicate liver, lung, and spleen DNA samples,respectively. The arrow designates a 3.4 kb fragment derived fromAv1ALAPH81 sequences. Lanes 4 and 5 represent liver DNA from anuninjected control mouse, and a mouse that received the control vector,Av1ALH9B, respectively. Lanes 1-3 contain digested Av1ALH81 viral DNA inamounts equivalent to 25, 10, and 1 vector copies per cell. DNA markers(Gibco-BRL, Gaithersburg, Md.) sizes are indicated in Kb. The relativetransduction efficiencies of the organs was determined by PhosphorImager quantitation.

As had been observed previously, liver was the most efficientlytransduced organ, with lung and spleen having approximately 10% and 1%of the vector copy number of liver, respectively (FIG. 48A; Smith etal., 1993).

For the RNAse protection analysis, the RNA quantities were adjustedrelative to the organ transduction efficiency. (FIG. 48B.)

5 μg of total liver RNA was used in each reaction. Lung and spleen RNAquantities were adjusted relative to the organ transduction efficiency.The lung RNA concentrations used for each reaction were: Lanes 9-12; 50μg, 35 μg, 35 μg, and 12 μg. The spleen RNA concentrations were: Lanes14-17; 150 μg, 100 μg, 70 μg, and 100 μg. The arrow labeled FVIIIdesignates the 212 nt human FVIII-specific protected probe fragment.Lanes 1, 8, 13, and 18, contain ³² P-labeled DNA molecular weightmarkers. Lane 2 contains undigested full-length probe. Lane 3 containsliver RNA from an Av1ALH9B-injected control mouse. Exposure shown is 20days with intensifying screens. The lower panel displays a separateRNAse protection assay using 10 μg of each RNA, and an anti-sense probeencoding a portion of the mouse β-actin cDNA. The arrow labeled Actindesignates the 210 nt mouse actin-specific protected probe fragment.Exposure shown is 6 hr with intensifying screen.

Using an anti-sense RNA probe complimentary to the FVIII coding region,a FVIII-specific protected fragment of the predicted size (212 nts; FIG.48C) was detected only in the liver RNA samples (FIG. 48B, lanes 4-7).No FVIII-specific RNA was found in the lung or spleen RNA samples (FIG.48B, lanes 9-12, and lanes 14-17), or in the Av1ALH9B-transduced controlliver RNA (FIG. 48B, lane 3). Analysis of equal quantities of RNA (10μg) from all samples with an anti-sense RNA probe specific to the mouseactin coding region verified the integrity of the RNA within each of thesamples (FIG. 48B, lower panel). Notably, the differences in the actinRNA levels observed in the liver, lung, and spleen samples probablyreflects the normal variation in the endogenous expression levels ofactin within the three organs. Therefore, the albumin promoterincorporated into an adenoviral vector directed the expression of BDDFVIII in a liver-specific manner.

Biological Activity of FVIII Produced in Vivo

Because the FVIII-encoding adenoviral vector, Av1ALAPH81, mediated theexpression of human FVIII in mice at 10-fold human physiological levels(FIG. 45), it was important to verify that the FVIII produced in themice was biologically active. Pooled plasma from groups of ten mice eachthat received Av1ALAPH81, Av1ALH9B, or no vector injection, was analyzedby ELISA to measure human FVIII antigen and Coatest to determine FVIIIbiological activity (both mouse and human; Table VI). In thisexperiment, groups of ten mice each received 4×10⁹ pfu of Av1ALAPH81(FVIII), Av1ALH9B (FIX), or no injection (Neg). Plasma was collected andpooled prior to injection (time 0) or at the indicated times postinjection, and analyzed by ELISA for human FVIII antigen and by Coatestfor FVIII (mouse and human) biological activity. The corrected Coatestactivity units were calculated by subtracting the FVIII activitymeasured in the FIX-transduced group from the activity detected in thepooled FVIII plasma samples. The corrected activity units were convertedto ng using the definition of one Coatest unit equal to the amount ofFVIII in one ml of human pooled plasma, 100 to 200 ng/ml (Vehar et al.,1991).

                  TABLE VI    ______________________________________            ELISA    Coatest FVIII Biological Activity    Pooled        Human FVIII                             Actual Corrected                                           Calculated    Plasma          Time    (ng/ml)    (mU)   (mU)   (ng)    ______________________________________    FVIII 0       0          2155   --     --    FIX           0          2108   --     --    Neg           0          2014   --     --    FVIII 1 week  1470       16214  10494  1049-2098    FIX           0          5720   --     --    Neg           0          2484   --     --    FVIII 2 weeks 1149       15033   7864   786-1572    FIX           0          7169   --     --    Neg           0          2431   --     --    ______________________________________

The endogenous mouse FVIII activity, determined for all groups prior tovector administration, was found to be approximately 2000 mU/ml (TableVI). Notably, the mice that received the control vector, Av1ALH9B,showed a two to three-fold increase in endogenous mouse FVIII levels, asno human FVIII was detected in the pooled plasma as determined by ELISA(Table VI). Therefore, to determine the amount of human FVIII in themice that received Av1ALAPH81 above the endogenous mouse FVIII levels,the FVIII activities measured in the control vector-transduced groupwere subtracted from the activity detected in the pooled Av1ALAPH81plasma samples, to give the corrected mU/ml levels. At both one and twoweeks post injection, the amount of human FVIII calculated from thecorrected activity values matched the human FVIII antigen levels asdetermined by ELISA. Therefore, these data indicate that the high levelof human FVIII produced in the mice was biologically active.

High level FVIII Expression in Vivo with Administration of a Low VectorDose

Because administration of a high dose, 4×10⁹ pfu, of Av1ALAPH81 to miceresulted in expression of FVIII in the mouse plasma at ten-fold humanphysiological levels (FIG. 45), it was feasible that injection ofsignificantly lower vector doses to mice may allow expression oftherapeutic levels of FVIII. Therefore, groups of five mice each wereinjected intravenously with 5×10⁸ pfu of Av1ALAPH81 or Av1ALH81, andplasma FVIII levels were determined by ELISA weekly for four weeks (FIG.49). One week post injection, human FVIII levels in the plasma of micethat had received Av1ALAPH81 averaged 257±120 ng/ml. At four weeks,these levels had increased to 397±78 ng/ml. However, mice that hadreceived an identical dose of Av1ALH81 did not produce detectable levelsof FVIII (FIG. 49). In addition, no FVIII was detected in mice thatreceived the control vector, Av1ALH9B, or in uninjected control mice(data not shown). Therefore, administration of an eight-fold lower doseof Av1ALAPH81 to mice allowed the expression of human FVIII in the miceat two to three-fold human physiological levels.

EXAMPLE 14 Sustained Expression of Physiological Levels of FunctionalHuman Factor VIII in Mice

It was shown that intravenous administration of a high dose, 4×10⁹ pfu,of Av1ALAPH81 (FIG. 23) to mice mediated the expression of human FVIIIin the mouse plasma in amounts 10-fold higher than human physiologicallevels (>1000 ng/ml one week post injection) (Connelly et al., 1995) Todetermine if high level human FVIII expression could be achieved withadministration of lower vector doses, 5 groups of 5 mice each wereinjected, via tail vein, with decreasing doses of Av1ALAPH81, in amountsof 4×10⁹ ; 5×10⁸ ; 2×10⁸ ; and 5×10⁷ pfu/mouse. These amounts areequivalent approximately to 1.6×10¹¹ pfu/kg; 2.0×10¹⁰ pfu/kg; 8.0×10⁹pfu/kg; and 2.0×10⁹ pfu/kg, respectively. Plasma levels of human FVIIIwere measured by a human FVIII-specific ELISA (Connelly et al., 1995) atthe indicated times ranging from one to 22 weeks post vectoradministration (FIG. 50). At weekly intervals, plasma samples werecollected and human Factor VIII antigen was quantitated by ELISA. Datain FIGS. 50A and 50B are plotted as a mean value and standard deviationat each time point. Mice that received the highest dose of vector, 4×10⁹pfu, showed extremely high levels of FVIII expression, peaking threeweeks post injection at 2063±446 ng/ml (FIG. 50A). With administrationof 1×10⁹ pfu of Av1ALAPH81 to mice, peak plasma human FVIII expressionwas 440±80 ng/ml at two weeks. However, in both groups, a sharp declinein FVIII expression was detected beginning at 4-6 weeks, with plasmaFVIII levels dropping below the limit of sensitivity of the assay (60ng/ml) by 8-10 weeks (FIG. 50A). Alternatively, a different pattern ofFVIII expression was observed in mice that received the lower vectordoses (FIG. 50B). At a dose of 5×10⁸ pfu, FVIII expression peaked at397±78 ng/ml four weeks post injection. Peak expression levels at dosesof 2×10⁸ pfu and 5×10⁷ pfu were 168±61 ng/ml at 8 weeks, and 75±63 ng/mlat 10 weeks after vector administration, respectively. However, by 17 to22 weeks human FVIII was undetectable in the mouse plasma (FIG. 50B). Nohuman FVIII was detected in four mice that received 4×10⁹ pfu ofAv1ALH9B, a recombinant adenovirus encoding the human factor IX (FIX)cDNA (Connelly et al., 1995a; 1995b), or in uninjected control mice(data not shown). It is noteworthy that at 15 weeks at least one mousewithin each of the low dose groups were expressing human FVIII at humanphysiological levels (>100 ng/ml). Interestingly, mice that receivedeither of the two lowest vector doses showed a delay in the onset ofFVIII expression (FIG. 50B). Therefore, at lower vector doses, peakFVIII expression was delayed and FVIII expression showed a gradualdecline over 15 to 22 weeks (FIG. 50B), compared to the sharp drop inFVIII expression between 4 to 6 weeks observed with the high vectordoses (FIG. 50A). These results reveal that administration of low vectordoses allowed the sustained expression of physiological levels of humanFVIII in mice for at least 15 weeks.

The loss in detectable levels of FVIII in the mouse plasma 17 to 22weeks after vector administration could be caused by several factors.For example, vector DNA may have been eliminated from the liver, thealbumin promoter could have become inactivated, or the mice may havedeveloped an antibody response directed against human FVIII. Todistinguish between these possibilities, mouse liver DNA and RNAobtained from mice injected with 4×10⁹ pfu or 5×10⁸ pfu of Av1ALAPH81one and 22 weeks after vector administration was analyzed (FIG. 51).Groups of mice that received a dose of 4×10⁹ pfu or 5×10⁸ pfu ofAv1ALAPH81 were sacrificed at one week or at 22 weeks after vectoradministration. Liver DNA and RNA were isolated from each mouse liver.FIG. 51A shows Southern analysis of mouse liver DNA isolated from micethat received 4×10⁹ pfu of Av1ALAPH81. Each DNA sample was digested withBamHI, and subjected to Southern analysis. The arrow designates a 3.4 kbfragment containing Av1ALAPH81-derived Factor VIII sequences. Thestandards were generated by digesting purified Av1ALAPH81 viral DNA inamounts equivalent to 25, 10, and 1 vector copies per cell. DNA markersizes are indicated in kb. For mice that received the high vector dose,Southern analysis of liver DNA with comparison to vector copy numberstandards of 25, 10, and 1 copies (FIG. 51A, lanes 1-3) showed anaverage of 45 vector copies per cell, at one week post injection (FIG.51A, lanes 6-9), compared to an average of 0.2 copies per cell at 22weeks (FIG. 51A, lanes 10-13) revealing that the majority of vector DNAhad been lost from the mouse livers. No FVIII-containing vector wasdetected in an uninjected control mouse liver DNA sample (FIG. 51A, lane4), or a liver DNA sample isolated from a mouse injected with 4×10⁹ pfuof Av1ALAPH81 (FIG. 51A, lane 5).

The relative levels of human FVIII RNA produced in the mice thatreceived the high vector dose was determined by RNAse protectionanalysis using RNA isolated from the mouse livers (FIG. 51B), and ananti-sense RNA probe encoding a portion of the FVIII coding region(Connelly et al., 1995b). 50 μg of total cellular RNA isolated from themouse liver were used in each reaction. The arrow labeled FVIIIdesignates the 212 nt human Factor VIII-specific probe fragment. Thelanes marked Neg contain RNA isolated from an uninjected control mouse,and a mouse that received a similar dose of Av1ALH9B, a Factor IXencoding adenoviral vector. The lane marked P contains undigestedfull-length probe. The lane marked M contains ³² P-labeled DNA molecularweight markers. The lower panel displays a separate RNase protectionassay using 20 μg of total cellular mouse liver RNA, and an antisenseRNA probe encoding a portion of the mouse glyceraldehyde3-phosphodehydrogenase (GAPDH) cDNA. The arrow labeled GAPDH designatesthe 134 nt mouse GAPDH-specific protected probe fragment. With theFactor VIII-specific probe fragment, human FVIII-specific mRNA shouldprotect a 212 nt fragment. A high level of human FVIII-specific RNA wasdetected with the RNA samples isolated one week post injection (FIG.51B, lanes 4-7). However, by 22 weeks, human FVIII-specific RNA wasundetectable (FIG. 51B, lanes 8-11). No human FVIII-specific protectedprobe fragment was detected in the uninjected negative control RNAsample (FIG. 51B, lane 2) or with the Av1ALH9B-injected control mouseliver sample (FIG. 51B, lane 3). As an internal RNA standard, the mouseliver RNA samples were analyzed in a separate RNAse protection assayusing an antisense RNA probe containing a portion of the mouseglyceraldehyde-3-phosphate dehydrogenase (GAPDH) coding region (FIG.51B, lower panel). Therefore, the dramatic loss of vector DNA and,concordantly, human FVIII-specific RNA from the livers of mice thatreceived the high vector dose parallels the rapid drop in detectablelevels of human FVIII in the mouse plasma.

FIG. 51C depicts the Southern analysis of mouse liver DNA isolated frommice that received 5×10⁸ pfu of Av1ALAPH81. Each DNA was digested withBamHI, and subjected to Southern analysis. The arrow designates a 3.4 kbfragment containing Av1ALAPH81-derived Factor VIII sequences. Thestandards were generated by digesting purified Av1ALAPH81 viral DNA inamounts equivalent to 25, 10, and 1 vector copies per cell. DNA sizemarkers are indicated in kb.

In contrast, Southern analysis of DNA isolated from the livers of micethat received the low, 5×10⁸ pfu dose of Av1ALAPH81 (FIG. 51C), revealedthat at one week post injection the mice had an average of 7 vectorcopies per cell (FIG. 51C, lanes 6-10). At 22 weeks, however, the micehad retained an average of 0.5 vector copies per cell (FIG. 51C, lanes11-15). No vector was detected in an uninjected control mouse DNA sample(FIG. 51C, lane 4), or in a liver DNA sample derived from a mouseinjected with Av1ALH9B (FIG. 51C, lane 5). Therefore, mice that receivedthe low vector dose showed only a 14-fold drop in liver vector DNAlevels, compared to the dramatic 225-fold decrease in vector copy numberobserved in the mice that had received the high vector dose.

RNAse protection analysis using RNA isolated from the mouse livers (FIG.51D), and the human FVIII-specific anti-sense RNA probe revealed a highlevel of FVIII-specific mRNA at both one and 22 weeks after vectoradministration. 50 μg of total cellular RNA isolated from livers of micewhich received 5×10⁸ pfu of Av1ALAPH81 were used in each reaction. Thearrow labeled FVIII designates the 212 nt human Factor VIII specificprotected probe fragment. The lanes marked Neg contain RNA isolated froman uninjected control mouse, and a mouse that received a similar dose ofAv1ALH9B. The lane marked P contains undigested full-length probe. Thelane marked M contains ³² P-labeled DNA molecular weight markers. Thelower panel displays a separate RNase protection assay using 20 μg oftotal cellular mouse liver RNA, and an antisense RNA probe encoding aportion of the mouse GAPDH cDNA. The arrow labeled GAPDH designates the134 nt mouse GAPDH-specific protected fragment. Quantitation byphosphorimager scanning showed only a 3-fold decrease in the FVIII mRNAlevels from 1 to 22 weeks. No human FVIII-specific protected probefragment was detected in the uninjected negative control RNA sample(FIG. 51D, lane 2) or with the Av1ALH9B-injected control mouse liversample (FIG. 51D, lane 3). As an internal RNA standard, the mouse liverRNA samples were analyzed in a separate RNAse protection assay using themouse GAPDH anti-sense RNA probe (FIG. 51D, lower panel). Since asignificant amount of vector DNA remained in the mouse livers, and ahigh level of FVIII-specific RNA was detected at 22 weeks after vectoradministration, it is probable that loss of FVIII expression by 22 weekswas not due to a loss of vector DNA from the mouse livers, or to albuminpromoter inactivation.

Hepatotoxicity in Mice that Received High or Low Vector Doses

In another experiment, purified full-length human Factor VIII proteinwas administered to 3 mice that had been treated 4 months earlier with5×10⁸ pfu of Av1ALAPH81, and to 3 untreated mice. Plasma samples wereobtained from each mouse at 1 hr., 2 hrs., 4 hrs., 8 hrs., and 12 hrs.after administration, and assayed for human Factor VIII by ELISA. Thedata are plotted in FIG. 52 as a mean value and standard deviation ateach time point.

Since lower vector doses (5×10⁸ pfu and below) allow sustainedexpression of human FVIII in mice (FIG. 50), due, at least in part, tothe retention of vector DNA in the mouse livers (FIGS. 51 and 52), it isprobable that lower vector doses are less hepatoxic than higher doses,thus decreasing the turn-over of transduced hepatocytes. To showdirectly that a high dose of Av1ALAPH81 is more toxic than a lower dose,groups of 10 mice each were injected with a high dose (4×10⁹ pfu) or alow dose (5×10⁸ pfu) of Av1ALAPH81, injection medium only, oruninjected. Serum samples were collected prior to vector administration,and at the indicated times post injection, and analyzed for the presenceof four liver enzymes: aspartate aminotransferase (AST), alanineaminotransferase (ALT), sorbital dehydrogenase (SDH), and alkalinephosphatase (Alk Φ) (FIG. 53). FIG. 53A depicts aspartateaminotransferase (AST) levels. FIG. 53B depicts alanine aminotransferase(ALT) levels. FIG. 53C shows sorbitol dehydrogenase (SDH) levels. FIG.53D shows alkaline phosphatase levels. A dramatic increase in the levelsof all four liver enzymes was detected in the mice that received thehighest dose of vector. This increase persisted for at least four weekspost injection. The lower vector dose caused a slight increase in allliver enzymes above the levels of the control groups, or thepreinjection samples. Interestingly, by 12 weeks, both vector groupsshowed enzyme levels similar to the preinjection values and those of thecontrol group. In addition, at the 20 hr, 1 week, and 4 week time pointstwo mice from each group were sacrificed and liver sections analyzed byH&E staining. Liver sections from mice that received a high vector doseshowed a liver pathology including loss of the normal sinusodial liverarchitecture, lymphocytic infiltrate, and the presence of mitoticfigures. (personal communication, W. Hall, Pathology Associates Inc.,Frederick, Md.; data not shown). However, mice that received the lowvector dose looked morphologically normal (personal communication, W.Hall, Pathology Associates, Inc., Frederick, Md.; data not shown).Therefore, these results clearly indicate that a vector dose of 5×10⁸pfu of Av1ALAPH81 was significantly less hepatotoxic than an 8-foldhigher vector dose.

EXAMPLE 15 Treatment of Dog with Av1ALAPH81

A Factor VIII adenoviral vector, Av1ALAPH81 (FIG. 23), was used to treata Factor FVIII-deficient dog. Two vector lots were prepared (MT2-2 andMT2-3), titered on 293 cells, tested for the presence of RCA(replication competent adenovirus) by PCR analysis directed at E1asequences, and tested in mice before being used for treatment of thedog. In addition, prior to treatment, samples of the dog's plasma andserum were collected. Plasma samples from the Factor VIII-deficient dogand a normal dog, were tested in the human FVIII specific ELISA(Connelly et al., 1995) to look for cross reactivity of dog FVIII withthe human protein. Full-length recombinant FVIII protein generouslysupplied by Genetics Institute (Cambridge, Mass.) was used to generate astandard curve ranging from 1 to 100 ng/ml. BDD FVIII protein (suppliedby Genetics Institute, Cambridge, Mass.) and full-length recombinantFVIII were similarly quantified by this ELISA. Normal dog plasma did notinterfere with the assay and the limit of sensitivity with dog plasmasamples containing BDD FVIII was 3 to 6 ng/ml. Plasma samples werenormally diluted 1:10 for the ELISA. No cross reactivity of the ELISAwas found with the normal dog plasma at a 1:10 dilution, and it wasverified that the dog's plasma did not cross-react in the ELISA. Plasmafrom a normal dog and the Factor VIII-deficient dog was analyzed alsousing the Coatest chromogenic bio-assay (Chromogenix, Molndal, Sweden).Coatest measures the FVIII-dependent generation of Factor Xa from FactorX, with one unit defined as the amount of FVIII activity in one ml ofpooled human plasma, 100 to 200 ng/ml (Vehar et al., 1991). Pooled humanplasma (George King BioMedical, Inc., Overland Park, Kans.) was used asthe FVIII activity standard. It was confirmed that the dog wasFVIII-deficient, as no FVIII biological activity in his plasma wasdetected by Coatest. In addition, the canine FVIII levels weredetermined in the normal dog plasma to be 5,000 mU/ml, five times thelevel detected in normal human plasma. To look for the presence ofFVIII-inhibitory antibodies, a Coatest assay was performed in whichvarying amounts of human FVIII protein was added to the dog's plasmasamples. If the dog's plasma contained FVIII inhibitory antibodies, itwould be expected that FVIII biological activity to be inhibited andtherefore, not measurable in the assay. No evidence of FVIII inhibitoryantibodies was detected. Finally, the dog's serum was used in ananti-adenovirus antibody assay, to look for the presence of antibodiesspecific for human Ad5. None were detected.

Administration of Av1ALAPH81

Av1ALAPH81 (1×10¹² pfu) was administered to the dog, by cephalic veininjection. At days one through seven after vector administration, plasmaand serum samples were collected, and analyzed in the following manner:Clinical blood clotting tests: activated clotting time (ACT), andactivated partial thromboplastin time (APTT); Coatest assay to measureFVIII biological activity, and FVIII-specific ELISA, to look for thepresence of human FVIII. Cuticle bleed time was measured preinjection,and at days 2 to 7 after treatment. The activated clotting timeexperiments employed the Vacutainer® sterile evacuated glass tube andthe experiments were conducted according to the procedure contained inVacutainer® Brand Sterile Evacuated Glass Tube, Reorder No. 6522(Becton, Dickinson and Company, Rutherford, N.J.) (August 1992). TheAPTT was measured in accordance with Coles, Veterinary ClinicalPathology, W. B. Saunders Co., Philadelphia, pg. 106 (1986). Cuticlebleed time was measured in accordance with the procedure described inGiles, et al., Blood, Vol. 60, No. 3, pgs. 727-730 (September 1982).

Clinical Clotting Parameter Analyses

The clinical clotting tests, cuticle bleed time, ACT, and APTT weremeasured prior to vector administration and at several points aftertreatment. Prior to vector administration, the dog's cuticle bleed timewas measured to be greater than 20 minutes, indicative of thehemophiliac phenotype. However, at day 2 after treatment, the dog'scuticle bleed time was reduced to 2.5 minutes. Clotting times under 5minutes are considered normal. Therefore, administration of Av1ALAPH81to the dog caused the correction of the animal's bleeding defect. By day6, however, the cuticle bleed time was measured to be 7 minutes,slightly above the normal levels.

Measurement of the ACT showed the dog's clotting time to be greater than5 minutes prior to vector treatment. Normal ACT in dogs is less than 2minutes. At days 2 through 5 ACT values were measured at under 2minutes, indicating correction of the clotting deficiency. However, bydays 6 and 7, the ACT levels had risen back to abnormal levels.

The APTT was measured prior to vector administration and found to begreater than 20 seconds, indicative of a clotting deficiency. Days 2through 5 after vector treatment, the APTT was measured at or slightlyabove 10 seconds, indicating normal clotting. However, by days 6 to 8the APTT values were again measured at abnormal levels.

Measurement of FVIII Expression in Vector-Treated Dog

Plasma samples were analyzed using the Coatest assay for FVIIIbiological activity prior to vector treatment and daily for seven daysafter vector administration. Biologically active human FVIII wasmeasured using the Coatest bio-assay. Analysis of a plasma samplecollected prior to Av1ALAPH81 treatment indicated that the dog did notcontain detectable levels of functional FVIII. (FIG. 54.) However, atdays 1 to 5 after treatment, as shown in FIG. 54, plasma FVIII activityshowed a dramatic increase. FVIII expression peaked 2 days aftertreatment at greater than 8000 mU/ml. These levels are eight-fold higherthan the FVIII levels found in normal human plasma. Comparison of thepeak FVIII expression level to that of normal dog plasma revealed thatthe dog was producing FVIII at levels well above that detected in normaldog plasma. These data verify that correction of the clinical clottingparameters, i.e., cuticle bleed time, ACT, and APTT, was due to theproduction of functional FVIII. In addition, these results show thefirst example of phenotypic correction of the FVIII deficiency in aclinically relevant animal model. However, by days 6 and 7 FVIII plasmalevels were undetectable.

To verify that the FVIII expression measured in the dog's plasma was adirect result of vector administration, i.e., human FVIII, plasmasamples were assayed for the presence of human FVIII by ELISA. No humanFVIII was measured in the dog's plasma prior to vector administration.At days 2 through 5 high levels of human FVIII were measured, as shownin FIG. 55, verifying that all the FVIII measured in the dog plasma wasvector-derived. Conversion of the Coatest activity units to ng/ml usingthe definition: one unit corresponds to the amount of FVIII in one ml ofhuman pooled plasma, 100 ng/ml, and comparison of human FVIII expressionlevels detected by ELISA, revealed that similar levels of FVIII weredetected by both assays. This observation confirms the accuracy andvalidity of the human FVIII-specific ELISA.

Analysis of Liver and Spleen Biopsy Samples

To determine if the decline in FVIII expression levels in the dog'splasma by days 5 to 8 after vector administration was due to loss ofvector DNA from the liver or due to transcriptional inactivation of thealbumin promoter, an open biopsy was performed 8 days after treatment.DNA and RNA were isolated from liver and spleen samples collected duringthe biopsy, and used in Southern and RNAse Protection analyses. DNA wasisolated from liver and spleen biopsy samples using standard procedures.Briefly, organ sections were minced and incubated overnight inSDS/Proteinase K buffer. This was followed by three phenol/chloroformextractions, one chloroform extraction, ethanol precipitation andresuspension in water. 20 μg of each DNA sample were digested with BamHI and subjected to Southern analysis, results of which are shown inFIG. 56. The probe, prepared by random oligonucleotide priming,contained human FVIII cDNA sequences from +73 to +1345 (Toole et al.,1984; Wood et al., 1984). The copy number control standards wereprepared by adding 1.2 ng, 120 pg or 12 pg of viral DNA, equivalent to10, 1, and 0.1 vector copies per cell, respectively, to 20 μg of normaldog control liver genomic DNA and digesting with BamHI. The bandintensities were quantitated with a Molecular Dynamics PhosphorImagerSF. A high level of vector-specific DNA was detected in the liver andspleen biopsy samples, approximately 5 and 10 vector copies per cell,respectively. No vector DNA was observed in a liver biopsy samplecollected from a normal dog, or from a pre-injection liver biopsy samplecollected from the dog.

To perform the RNA analysis, RNA was isolated from biopsy samples usingthe RNAzole B (Tel-Test, Friendswood, Tex.) extraction method. RNAseprotection analyses were performed using the RNAse Protection Kit II(Ambion, Austin, Tex.). For each sample, quantities of 50 μg of totalcellular RNA were hybridized for 12 hrs at 45° C. with 5×10⁴ cpm of agel-purified RNA probe (See below.), digested with the RNAse A/T1solution provided with the kit, diluted 1:100, processed as directed,and analyzed on an 8% polyacrylamide-8 M urea gel (SequaGel, NationalDiagnostics, Atlanta, Ga.). The band intensities were quantitated with aMolecular Dynamics PhosphorImager SF. The FVIII probe template,pGemSRpr, was constructed by inserting the 204 bp Sac I-Eco RI fragmentisolated from pMT2LA (provided by Genetics Institute, Cambridge, Mass.)(Toole et al., 1986) into pGem4Z (Promega, Madison, Wis.) cut with Sac Iand Eco RI. The RNA analysis revealed the presence of a high level ofFVIII-specific RNA only in the liver biopsy sample. (FIG. 56.) NoFVIII-specific RNA was detected in the spleen biopsy sample, thepreinjection liver sample, or in the normal dog liver RNA sample. Toverify the integrity of the RNA in each sample, a separate RNAseprotection analysis was performed using a mouse-specific glyceraldehyde3-phosphate dehydrogenase (GAPDH) probe. The GAPDH probe template wasgenerated from the pTRI-GAPDH mouse plasmid (Ambion, Austin, Tex.)digested with Sty I. A similar amount of RNA was detected in each of theliver RNA samples. However, less GAPDH-specific RNA was detected in thespleen-derived RNA sample, probably reflecting the normal variation inGAPDH levels between different organs. These results indicate that thealbumin promoter remained transcriptionally active 8 days after vectoradministration. In addition, consistent with previous observations inmice, these data reveal that the albumin promoter functioned in aliver-specific manner in the dog, as no FVIII-specific RNA was detectedin the spleen biopsy sample although the spleen contained more vectorcopies per cell than the liver. Taken together, these results indicatethat the loss of FVIII expression by 8 days after treatment was not dueto a complete loss of vector DNA from the treated animal, nor to thetranscriptional inactivation of the albumin promoter. Previous studieshave shown that FVIII-deficient dogs have a high likelihood ofdeveloping human FVIII inhibitory antibodies, (Littlewood andBarrowcliffe, 1987; Thrombosis and Haemostasis, Vol. 57, pages 314-321).Therefore, the drop in FVIII plasma levels between 5 to 8 days aftertreatment may have been due to the presence of FVIII inhibitoryantibodies in the dog's plasma.

EXAMPLE 16 Generation of Human Factor VIII-Encoding RecombinantAdenoviruses Av1AP3'H81, Av1ALAP3'H81, Av1APH8H9, and Av1ALAPH8H9Construction of Av1AP3'H81 and Av1ALAP3'H81

The adenoviral shuttle plasmid, pAvAP3'H81 was constructed by replacingthe FVIII 3' UTR (215 bp) in the plasmid, pAvAPH81 (FIG. 18), with theApo A1 3' UTR and poly(A) signal (Genbank #XO7496, nts 2024 to 2143),and used to generate the recombinant adenoviral vector, Av1AP3'H81 byprocedures hereinabove described. The shuttle plasmid, pAvALAP3'H81, wasconstructed by replacing the FVIII 3' UTR (215 bp) in the plasmid,pAvALAPH81 (FIG. 20) with the Apo A1 sequences described above and usedto generate the recombinant adenoviral vector, Av1ALAP3'H81 (FIG. 57) byprocedures hereinabove described.

Construction of Av1APH8H9 and Av1ALAPH8H9

The shuttle plasmid, pAvAPH8H9 was constructed by replacing the FVIII 3'UTR (215 bp) in the plasmid, pAvAPH81 (FIG. 18), with the 3' UTR andpoly(A) signals of the human FIX gene (1.7 kb). In a similar manner,pAvALAPH8H9 was constructed by replacing the FVIII 3' UTR (215 bp) inpAvALAPH81 (FIG. 20) with the FIX 3' sequences. The plasmid, pAvAPH8H9was used to generate Av1APH8H9 by procedures hereinabove described.Similarly, the plasmid, pAvALAPH8H9, was used to generate Av1ALAPH8H9.(FIG. 57). However, instead of using the large Cla I fragment isolatedfrom dl327, the large Cla I fragment was isolated from dl7001 (Ranheimet al., 1993; Journal of Virology, Vol. 67, pages 2159-2167), and usedin the cotransfection of 293 cells as described. It was necessary to usedl7001 instead of dl327 because dl7001 contains a larger deletion of theE3 region than does dl327. Due to the large size of the shuttle plasmid,pAvALAPH8H9, it was necessary to use dl7001 to generate a stablerecombinant adenoviral vector.

In Vitro Analysis of FVIII Adenoviral Vectors

To determine if the FVIII adenoviral vectors produced functional FVIIIin vitro, HepG2, human hepatoma cells, were transduced with each FVIIIadenoviral vector, Av1ALAPH81, Av1ALAP3'H81, Av1APH81 (FIG. 23),Av1AP3'H81, Av1APH8H9, or Av1ALH9B (an albumin promoted human FIXadenoviral vector used as a negative control), at an MOI of 30, intriplicate infections. Also, triplicate wells of a six-well plate werealso mock infected. 12 hours after transduction, media was changed, and24 hours later, media was assayed for the presence of functional FVIIIby the Coatest assay. The data are plotted as a mean value and standarddeviation. The results of two separate experiments (FIG. 58) aredisplayed, and reveal that all FVIII vectors produce high levels offunctional FVIII in vitro.

Analysis of FVIII Adenoviral Viral Vectors in Mice

To determine if the FVIII adenoviral vectors produced human FVIII whenadministered to mice, a dose of 4×10⁹ pfu of Av1ALAP3'H81, Av1APH81,Av1AP3'FH81, Av1APH8H9 or Av1ALH9B was administered to groups of fivemice each. Expression from the new vectors was compared to that obtainedwith a group of five mice that had received a dose of 5×10⁸ pfu ofAv1ALAPH81. One week after vector administration, human FVIII plasmalevels in the mice were measured by ELISA. Data are shown as a meanvalue and standard deviation. (FIG. 59.) The data reveal that allvectors are functional in vivo. However, expression in mice thatreceived a high dose of Av1APH81, Av1AP3'H81, and Av1APH8H9 wassignificantly lower than that detected from mice injected with a lowdose of Av1ALAPH81. In addition, expression from mice injected with ahigh dose of Av1ALAP3'H81 was higher than that detected in mice injectedwith a low dose of Av1ALAPH81. However, injection of 4×10⁹ pfu ofAv1ALAPH81 to mice resulted in expression of 1000 to 2000 ng/ml of humanFVIII (data not shown), significantly higher than that detected in micethat received a similar dose of Av1ALAP3'H81 (400 ng/ml).

EXAMPLE 17 Generation of Factor IX Adenoviral Vectors Having a Deletedor Mutated Tripartite Leader (TPL) Sequence

The adenoviral vector Av1H9FR was reconstructed to remove an openreading frame (orf) in the tripartite leader (TPL) between the RSVpromoter and the Factor IX cDNA. FIG. 60 shows a diagram of the left endof Av1H9FR. The vector begins with natural adenovirus serotype 5 (Ad5)sequences, starting with the inverted terminal repeat (ITR) followed bythe packaging signal (ψ) and the E1a enhancer (E1a enh). This isfollowed by the RSV promoter and the Ad5 TPL. Downstream of the TPL isthe human Factor IX cDNA (huFIX). In Av1H9B, Av1H9D, Av1H9ER, andAv1H9FR, an ATG in the context of a reasonably good Kozak consensussequence is situated immediately upstream of the TPL. A 63 amino acidopen reading frame (orf) follows the ATG. Translation initiation at thisATG would likely have a strong deleterious effect on translation of theFactor IX cDNA.

Two new vectors were generated. The first one, Av1H9F1, lacks the entireTPL, including the orf. The second vector, Av1H9F2, retains the TPL;however, the ATG at the beginning of the TPL was replaced with a CTG.

The first step in the construction of Av1H9F1 was to delete the TPL fromthe shuttle plasmid pAv1H9FR (FIG. 39). This was accomplished bydigesting the plasmid with the restriction enzymes SfiI and SpeI. Theresulting DNA fragments were subjected to electrophoresis in an agarosegel and the larger of the two fragments was recovered by electroelution.The ends of the DNA were made blunt by treatment with T4 DNA Polymerase,then the fragment was circularized by ligation. An aliquot of theligation mixture was used to transform competent DH5 E. coli, andampicillin-resistant colonies were isolated. Several colonies wereamplified and miniprep DNA was analyzed by restriction enzyme digestion.A clone with the correct restriction pattern was identified andexpanded. The resulting shuttle plasmid, pAvS15H9F, was co-transfectedwith the large DNA fragment of ClaI digested Ad-dl327 into 293 cells.Two weeks later, infectious recombinant adenoviral vector plaques werepicked, expanded, and screened for expression of Factor IX by ELISA. Onepositive clone was purified by isolating a single plaque, thenamplified. The resulting recombinant adenoviral vector was calledAv1H9F1. Its integrity was verified by restriction enzyme diagnostics.The structure of the left end of this vector is shown in FIG. 60. Theextreme left end of the vector contains the normal sequence ofadenovirus serotype 5 (Ad5), including the inverted terminal repeat(ITR). This region is followed by the RSV promoter, which is immediatelyfollowed by the human Factor IX cDNA. The ATG shown in the schematic forAv1H9F1 represents the Factor IX start codon.

To construct Av1H9F2, the shuttle plasmid pAv1H9FR was digested with therestriction enzyme SfiI, the DNA ends were made blunt using T4 DNAPolymerase, and the DNA molecule was recircularized by ligation.Competent DH5 cells were transformed and ampicillin-resistant cloneswere amplified and screened by restriction enzyme digestion of miniprepDNA. A positive clone was identified and the resulting shuttle plasmidwas referred to as pAvS17H9F.

Subsequently, 293 cells were cotransfected with pAvS17H9F and the largeDNA fragment of ClaI digested Ad-dl327. Recombinant adenoviral vectorplaques were picked, expanded, and screened for expression of Factor IXby ELISA. A positive clone was identified and amplified, thus generatingthe vector Av1H9F2. A schematic of the left end of the vector is shownin FIG. 60. Av1H9F2 identical to Av1H9FR, except for a 5 base pairdeletion at the beginning of the TPL, which effectively changes the ATGinto a CTG. The structure of the vector was verified by restrictionenzyme diagnostics and by DNA sequence analysis of the region betweenthe RSV promoter and the 3' untranslated region of the Factor IX cDNA.

The parent vector, Av1H9FR, and the two new vectors, Av1H9F1 andAv1H9F2, were compared in mice for their ability to mediate expressionof human Factor IX. Five mice received 5×10⁷ pfu and another 5 micereceived 1×10⁸ pfu of vector via tail vein injection. One week later,plasma samples were prepared and analyzed by ELISA for human Factor IX.The plasma level of Factor IX for each mouse is shown in Table VII.

                  TABLE VII    ______________________________________    Mouse      Vector     Dose    ng/ml huFIX    ______________________________________     1         Av1H9FR    1 × 10.sup.8                                  3155     2         Av1H9FR    1 × 10.sup.8                                  1723     3         Av1H9FR    1 × 10.sup.8                                  996     4         Av1H9FR    1 × 10.sup.8                                  574     5         Av1H9FR    1 × 10.sup.8                                  704     6         Av1H9FR    5 × 10.sup.7                                  93     7         Av1H9FR    5 × 10.sup.7                                  150     8         Av1H9FR    5 × 10.sup.7                                  153     9         Av1H9FR    5 × 10.sup.7                                  97    10         Av1H9F1    1 × 10.sup.8                                  427    11         Av1H9F1    1 × 10.sup.8                                  1840    12         Av1H9F1    1 × 10.sup.8                                  266    13         Av1H9F1    1 × 10.sup.8                                  1626    14         Av1H9F1    1 × 10.sup.8                                  331    15         Av1H9F1    5 × 10.sup.7                                  254    16         Av1H9F1    5 × 10.sup.7                                  129    17         Av1H9F1    5 × 10.sup.7                                  91    18         Av1H9F1    5 × 10.sup.7                                  113    19         Av1H9F1    5 × 10.sup.7                                  64    20         Av1H9F2    1 × 10.sup.8                                  14308    21         Av1H9F2    1 × 10.sup.8                                  20313    22         Av1H9F2    1 × 10.sup.8                                  10886    23         Av1H9F2    1 × 10.sup.8                                  5683    24         Av1H9F2    1 × 10.sup.8                                  5927    25         Av1H9F2    5 × 10.sup.7                                  1035    26         Av1H9F2    5 × 10.sup.7                                  867    27         Av1H9F2    5 × 10.sup.7                                  1755    28         Av1H9F2    5 × 10.sup.7                                  2164    29         Av1H9F2    5 × 10.sup.7                                  3220    ______________________________________

To normalize the expression levels to the vector content in the liver,the animals were sacrificed and DNA from their livers was analyzed bySouthern (FIG. 61). The results show that Av1H9F2 pressed approximately10-15 times higher levels of Factor IX than either Av1H9FR or Av1H9F1,even though the vector content in the liver was nearly the same for eachvector. The average level of Factor IX expression for each cohort ofmice is shown above the Southern blots.

All patents, publications, and database accession numbers, anddepository accession numbers referenced in this specification areindicative of the level of skill of persons in the art to which theinvention pertains. The disclosures of all such patents, publications(including published patent applications), and database accessionnumbers, and depository accession numbers are specifically incorporatedherein by reference in their entirety to the same extent as if each suchindividual patent, publication, and database accession number, anddepository accession number were specifically and individually indicatedto be incorporated by reference.

It is to be understood, however, that the scope of the present inventionis not to be limited to the specific embodiments described above. Theinvention may be practiced other than as particularly described andstill be within the scope of the accompanying claims.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES:  7    - (2) INFORMATION FOR SEQ ID NO: 1:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 32 bases              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: oligonucleotide    -     (ix) FEATURE:              (A) NAME/KEY: PCR prime - #r; oligo MGM8.293    #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #          32      TGAC CATGATTACG AA    - (2) INFORMATION FOR SEQ ID NO: 2:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 43 bases              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: oligonucleotide    -     (ix) FEATURE:              (A) NAME/KEY: PCR prime - #r; oligo MGM5.293    #2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    # 43               TGTC GACGCCGGAA AGGTGATCTG TGT    - (2) INFORMATION FOR SEQ ID NO: 3:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 38 bases              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: oligonucleotide    -     (ix) FEATURE:              (A) NAME/KEY: PCR prime - #r; oligo SSC 1.593    #3:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #     38           GTAC CCGGGAGACC TGCAAGCC    - (2) INFORMATION FOR SEQ ID NO: 4:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 45 bases              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: oligonucleotide    -     (ix) FEATURE:              (A) NAME/KEY: PCR prime - #r; oligo SSC 2.593    #4:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #45                TTGC ATCCTGAAGG GCCGTGGGGA CCTGG    - (2) INFORMATION FOR SEQ ID NO: 5:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 35 bases              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: oligonucleotide    -     (ix) FEATURE:              (A) NAME/KEY: PCR prime - #r; oligo SSC 3.593    #5:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #       35         ACTG CGAGAAGGAG GTGCG    - (2) INFORMATION FOR SEQ ID NO: 6:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 1548 bases              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: Factor IX - # cDNA    #6:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - AGGTTATGCA GCGCGTGAAC ATGATCATGG CAGAATCACC AGGCCTCATC AC - #CATCTGCC      60    - TTTTAGGATA TCTACTCAGT GCTGAATGTA CAGTTTTTCT TGATCATGAA AA - #CGCCAACA     120    - AAATTCTGAA TCGGCCAAAG AGGTATAATT CAGGTAAATT GGAAGAGTTT GT - #TCAAGGGA     180    - ACCTTGAGAG AGAATGTATG GAAGAAAAGT GTAGTTTTGA AGAAGCACGA GA - #AGTTTTTG     240    - AAAACACTGA AAGAACAACT GAATTTTGGA AGCAGTATGT TGATGGAGAT CA - #GTGTGAGT     300    - CCAATCCATG TTTAAATGGC GGCAGTTGCA AGGATGACAT TAATTCCTAT GA - #ATGTTGGT     360    - GTCCCTTTGG ATTTGAAGGA AAGAACTGTG AATTAGATGT AACATGTAAC AT - #TAAGAATG     420    - GCAGATGCGA GCAGTTTTGT AAAAATAGTG CTGATAACAA GGTGGTTTGC TC - #CTGTACTG     480    - AGGGATATCG ACTTGCAGAA AACCAGAAGT CCTGTGAACC AGCAGTGCCA TT - #TCCATGTG     540    - GAAGAGTTTC TGTTTCACAA ACTTCTAAGC TCACCCGTGC TGAGACTGTT TT - #TCCTGATG     600    - TGGACTATGT AAATTCTACT GAAGCTGAAA CCATTTTGGA TAACATCACT CA - #AAGCACCC     660    - AATCATTTAA TGACTTCACT CGGGTTGTTG GTGGAGAAGA TGCCAAACCA GG - #TCAATTCC     720    - CTTGGCAGGT TGTTTTGAAT GGTAAAGTTG ATGCATTCTG TGGAGGCTCT AT - #CGTTAATG     780    - AAAAATGGAT TGTAACTGCT GCCCACTGTG TTGAAACTGG TGTTAAAATT AC - #AGTTGTCG     840    - CAGGTGAACA TAATATTGAG GAGACAGAAC ATACAGAGCA AAAGCGAAAT GT - #GATTCGAA     900    - TTATTCCTCA CCACAACTAC AATGCAGCTA TTAATAAGTA CAACCATGAC AT - #TGCCCTTC     960    - TGGAACTGGA CGAACCCTTA GTGCTAAACA GCTACGTTAC ACCTATTTGC AT - #TGCTGACA    1020    - AGGAATACAC GAACATCTTC CTCAAATTTG GATCTGGCTA TGTAAGTGGC TG - #GGGAAGAG    1080    - TCTTCCACAA AGGGAGATCA GCTTTAGTTC TTCAGTACCT TAGAGTTCCA CT - #TGTTGACC    1140    - GAGCCACATG TCTTCGATCT ACAAAGTTCA CCATCTATAA CAACATGTTC TG - #TGCTGGCT    1200    - TCCATGAAGG AGGTAGAGAT TCATGTCAAG GAGATAGTGG GGGACCCCAT GT - #TACTGAAG    1260    - TGGAAGGGAC CAGTTTCTTA ACTGGAATTA TTAGCTGGGG TGAAGAGTGT GC - #AATGAAAG    1320    - GCAAATATGG AATATATACC AAGGTATCCC GGTATGTCAA CTGGATTAAG GA - #AAAAACAA    1380    - AGCTCACTTA ATGAAAGATG GATTTCCAAG GTTAATTCAT TGGAATTGAA AA - #TTAACAGG    1440    - GCCTCTCACT AACTAATCAC TTTCCCATCT TTTGTTAGAT TTGAATATAT AC - #ATTCTATG    1500    #              1548TTTA CAGGGGAGAA TTTCATATTT TACCTGAG    - (2) INFORMATION FOR SEQ ID NO: 7:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 4629 bases              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA primer    -     (ix) FEATURE:              (A) NAME/KEY: Factor VI - #II cDNA with B domain deleted    #7:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - ATGCAAATAG AGCTCTCCAC CTGCTTCTTT CTGTGCCTTT TGCGATTCTG CT - #TTAGTGCC      60    - ACAAGAAGAT ACTACCTGGG TGCAGTGGAA CTGTCATGGG ACTATATGCA AA - #GTGATCTC     120    - GGTGAGCTGC CTGTGGACGC AAGATTTCCT CCTAGAGTGC CAAAATCTTT TC - #CATTCAAC     180    - ACCTCAGTCG TGTACAAAAA GACTCTGTTT GTAGAATTCA CGGTTCACCT TT - #TCAACATC     240    - GCTAAGCCAA GGCCACCCTG GATGGGTCTG CTAGGTCCTA CCATCCAGGC TG - #AGGTTTAT     300    - GATACAGTGG TCATTACACT TAAGAACATG GCTTCCCATC CTGTCAGTCT TC - #ATGCTGTT     360    - GGTGTATCCT ACTGGAAAGC TTCTGAGGGA GCTGAATATG ATGATCAGAC CA - #GTCAAAGG     420    - GAGAAAGAAG ATGATAAAGT CTTCCCTGGT GGAAGCCATA CATATGTCTG GC - #AGGTCCTG     480    - AAAGAGAATG GTCCAATGGC CTCTGACCCA CTGTGCCTTA CCTACTCATA TC - #TTTCTCAT     540    - GTGGACCTGG TAAAAGACTT GAATTCAGGC CTCATTGGAG CCCTACTACT AT - #GTAGAGAA     600    - GGGAGTCTGG CCAAGGAAAA GACACAGACC TTGCACAAAT TTATACTACT TT - #TTGCTGTA     660    - TTTGATGAAG GGAAAAGTTG GCACTCAGAA ACAAAGAACT CCTTGATGCA GG - #ATAGGGAT     720    - GCTGCATCTG CTCGGGCCTG GCCTAAAATG CACACAGTCA ATGGTTATGT AA - #ACAGGTCT     780    - CTGCCAGGTC TGATTGGATG CCACAGGAAA TCAGTCTATT GGCATGTGAT TG - #GAATGGGC     840    - ACCACTCCTG AAGTGCACTC AATATTCCTC GAAGGTCACA CATTTCTTGT GA - #GGAACCAT     900    - CGCCAGGCGT CCTTGGAAAT CTCGCCAATA ACTTTCCTTA CTGCTCAAAC AC - #TCTTGATG     960    - GACCTTGGAC AGTTTCTACT GTTTTGTCAT ATCTCTTCCC ACCAACATGA TG - #GCATGGAA    1020    - GCTTATGTCA AAGTAGACAG CTGTCCAGAG GAACCCCAAC TACGAATGAA AA - #ATAATGAA    1080    - GAAGCGGAAG ACTATGATGA TGATCTTACT GATTCTGAAA TGGATGTGGT CA - #GGTTTGAT    1140    - GATGACAACT CTCCTTCCTT TATCCAAATT CGCTCAGTTG CCAAGAAGCA TC - #CTAAAACT    1200    - TGGGTACATT ACATTGCTGC TGAAGAGGAG GACTGGGACT ATGCTCCCTT AG - #TCCTCGCC    1260    - CCCGATGACA GAAGTTATAA AAGTCAATAT TTGAACAATG GCCCTCAGCG GA - #TTGGTAGG    1320    - AAGTACAAAA AAGTCCGATT TATGGCATAC ACAGATGAAA CCTTTAAGAC TC - #GTGAAGCT    1380    - ATTCAGCATG AATCAGGAAT CTTGGGACCT TTACTTTATG GGGAAGTTGG AG - #ACACACTG    1440    - TTGATTATAT TTAAGAATCA AGCAAGCAGA CCATATAACA TCTACCCTCA CG - #GAATCACT    1500    - GATGTCCGTC CTTTGTATTC AAGGAGATTA CCAAAAGGTG TAAAACATTT GA - #AGGATTTT    1560    - CCAATTCTGC CAGGAGAAAT ATTCAAATAT AAATGGACAG TGACTGTAGA AG - #ATGGGCCA    1620    - ACTAAATCAG ATCCTCGGTG CCTGACCCGC TATTACTCTA GTTTCGTTAA TA - #TGGAGAGA    1680    - GATCTAGCTT CAGGACTCAT TGGCCCTCTC CTCATCTGCT ACAAAGAATC TG - #TAGATCAA    1740    - AGAGGAAACC AGATAATGTC AGACAAGAGG AATGTCATCC TGTTTTCTGT AT - #TTGATGAG    1800    - AACCGAAGCT GGTACCTCAC AGAGAATATA CAACGCTTTC TCCCCAATCC AG - #CTGGAGTG    1860    - CAGCTTGAGG ATCCAGAGTT CCAAGCCTCC AACATCATGC ACAGCATCAA TG - #GCTATGTT    1920    - TTTGATAGTT TGCAGTTGTC AGTTTGTTTG CATGAGGTGG CATACTGGTA CA - #TTCTAAGC    1980    - ATTGGAGCAC AGACTGACTT CCTTTCTGTC TTCTTCTCTG GATATACCTT CA - #AACACAAA    2040    - ATGGTCTATG AAGACACACT CACCCTATTC CCATTCTCAG GAGAAACTGT CT - #TCATGTCG    2100    - ATGGAAAACC CAGGTCTATG GATTCTGGGG TGCCACAACT CAGACTTTCG GA - #ACAGAGGC    2160    - ATGACCGCCT TACTGAAGGT TTCTAGTTGT GACAAGAACA CTGGTGATTA TT - #ACGAGGAC    2220    - AGTTATGAAG ATATTTCAGC ATACTTGCTG AGTAAAAACA ATGCCATTGA AC - #CAAGAAGC    2280    - TTCTCCCAGA ATTCAAGACA CCCTAGCACT AGGCAAAAGC AATTTAATGC CA - #CCCCACCA    2340    - GTCTTGAAAC GCCATCAACG GGAAATAACT CGTACTACTC TTCAGTCAGA TC - #AAGAGGAA    2400    - ATTGACTATG ATGATACCAT ATCAGTTGAA ATGAAGAAGG AAGATTTTGA CA - #TTTATGAT    2460    - GAGGATGAAA ATCAGAGCCC CCGCAGCTTT CAAAAGAAAA CACGACACTA TT - #TTATTGCT    2520    - GCAGTGGAGA GGCTCTGGGA TTATGGGATG AGTAGCTCCC CACATGTTCT AA - #GAAACAGG    2580    - GCTCAGAGTG GCAGTGTCCC TCAGTTCAAG AAAGTTGTTT TCCAGGAATT TA - #CTGATGGC    2640    - TCCTTTACTC AGCCCTTATA CCGTGGAGAA CTAAATGAAC ATTTGGGACT CC - #TGGGGCCA    2700    - TATATAAGAG CAGAAGTTGA AGATAATATC ATGGTAACTT TCAGAAATCA GG - #CCTCTCGT    2760    - CCCTATTCCT TCTATTCTAG CCTTATTTCT TATGAGGAAG ATCAGAGGCA AG - #GAGCAGAA    2820    - CCTAGAAAAA ACTTTGTCAA GCCTAATGAA ACCAAAACTT ACTTTTGGAA AG - #TGCAACAT    2880    - CATATGGCAC CCACTAAAGA TGAGTTTGAC TGCAAAGCCT GGGCTTATTT CT - #CTGATGTT    2940    - GACCTGGAAA AAGATGTGCA CTCAGGCCTG ATTGGACCCC TTCTGGTCTG CC - #ACACTAAC    3000    - ACACTGAACC CTGCTCATGG GAGACAAGTG ACAGTACAGG AATTTGCTCT GT - #TTTTCACC    3060    - ATCTTTGATG AGACCAAAAG CTGGTACTTC ACTGAAAATA TGGAAAGAAA CT - #GCAGGGCT    3120    - CCCTGCAATA TCCAGATGGA AGATCCCACT TTTAAAGAGA ATTATCGCTT CC - #ATGCAATC    3180    - AATGGCTACA TAATGGATAC ACTACCTGGC TTAGTAATGG CTCAGGATCA AA - #GGATTCGA    3240    - TGGTATCTGC TCAGCATGGG CAGCAATGAA AACATCCATT CTATTCATTT CA - #GTGGACAT    3300    - GTGTTCACTG TACGAAAAAA AGAGGAGTAT AAAATGGCAC TGTACAATCT CT - #ATCCAGGT    3360    - GTTTTTGAGA CAGTGGAAAT GTTACCATCC AAAGCTGGAA TTTGGCGGGT GG - #AATGCCTT    3420    - ATTGGCGAGC ATCTACATGC TGGGATGAGC ACACTTTTTC TGGTGTACAG CA - #ATAAGTGT    3480    - CAGACTCCCC TGGGAATGGC TTCTGGACAC ATTAGAGATT TTCAGATTAC AG - #CTTCAGGA    3540    - CAATATGGAC AGTGGGCCCC AAAGCTGGCC AGACTTCATT ATTCCGGATC AA - #TCAATGCC    3600    - TGGAGCACCA AGGAGCCCTT TTCTTGGATC AAGGTGGATC TGTTGGCACC AA - #TGATTATT    3660    - CACGGCATCA AGACCCAGGG TGCCCGTCAG AAGTTCTCCA GCCTCTACAT CT - #CTCAGTTT    3720    - ATCATCATGT ATAGTCTTGA TGGGAAGAAG TGGCAGACTT ATCGAGGAAA TT - #CCACTGGA    3780    - ACCTTAATGG TCTTCTTTGG CAATGTGGAT TCATCTGGGA TAAAACACAA TA - #TTTTTAAC    3840    - CCTCCAATTA TTGCTCGATA CATCCGTTTG CACCCAACTC ATTATAGCAT TC - #GCAGCACT    3900    - CTTCGCATGG AGTTGATGGG CTGTGATTTA AATAGTTGCA GCATGCCATT GG - #GAATGGAG    3960    - AGTAAAGCAA TATCAGATGC ACAGATTACT GCTTCATCCT ACTTTACCAA TA - #TGTTTGCC    4020    - ACCTGGTCTC CTTCAAAAGC TCGACTTCAC CTCCAAGGGA GGAGTAATGC CT - #GGAGACCT    4080    - CAGGTGAATA ATCCAAAAGA GTGGCTGCAA GTGGACTTCC AGAAGACAAT GA - #AAGTCACA    4140    - GGAGTAACTA CTCAGGGAGT AAAATCTCTG CTTACCAGCA TGTATGTGAA GG - #AGTTCCTC    4200    - ATCTCCAGCA GTCAAGATGG CCATCAGTGG ACTCTCTTTT TTCAGAATGG CA - #AAGTAAAG    4260    - GTTTTTCAGG GAAATCAAGA CTCCTTCACA CCTGTGGTGA ACTCTCTAGA CC - #CACCGTTA    4320    - CTGACTCGCT ACCTTCGAAT TCACCCCCAG AGTTGGGTGC ACCAGATTGC CC - #TGAGGATG    4380    - GAGGTTCTGG GCTGCGAGGC ACAGGACCTC TACTGAGGGT GGCCACTGCA GC - #ACCTGCCA    4440    - CTGCCGTCAC CTCTCCCTCC TCAGCTCCAG GGCAGTGTCC CTCCCTGGCT TG - #CCTTCTAC    4500    - CTTTGTGCTA AATCCTAGCA GACACTGCCT TGAAGCCTCC TGAATTAACT AT - #CATCAGTC    4560    - CTGCATTTCT TTGGTGGGGG GCCAGGAGGG TGCATCCAAT TTAACTTAAC TC - #TTACCTAT    4620    #       4629    __________________________________________________________________________

What is claimed is:
 1. An adenoviral vector including at least one DNAsequence encoding a clotting factor.
 2. The vector of claim 1 whereinsaid DNA sequence encodes Factor VIII or a fragment thereof havingFactor VIII clotting activity.
 3. The vector of claim 2 wherein saidclotting factor is B domain deleted Factor VIII.
 4. The vector of claim3 wherein said vector further includes a tissue-specific promoter. 5.The vector of claim 4 wherein said tissue-specific promoter is the mousealbumin promoter.
 6. The vector of claim 5 wherein said vector furtherincludes at least one genomic element.
 7. The vector of claim 5 whereinsaid vector further includes the first exon of the apolipoprotein A-1gene, the first intron of the apolipoprotein A-1 gene, and the upstreamportion of the second exon of the apolipoprotein A-1 gene to the startcodon of the apolipoprotein A-1 gene.
 8. The vector of claim 3 whereinsaid vector further includes at least one genomic element.
 9. The vectorof claim 8 wherein said vector includes the ApoA1 promoter.
 10. Thevector of claim 9 wherein said vector further includes the first exon ofthe apolipoprotein A-1 gene, the first intron of the apolipoprotein A-1gene, and the upstream portion of the second exon of the apolipoproteinA-1 gene to the start codon of the apolipoprotein A-1 gene.
 11. Thevector of claim 9 wherein said vector further includes the first exonand first intron of the apolipoprotein A-1 gene.
 12. The vector of claim3 wherein said B domain deleted Factor VIII is human B domain deletedFactor VIII.
 13. The vector of claim 2 wherein said Factor VIII or afragment thereof is human Factor VIII or a fragment thereof havingFactor VIII clotting activity.
 14. The vector of claim 1 wherein saidDNA sequence encodes Factor IX or a fragment thereof having Factor IXclotting activity.
 15. The vector of claim 14 wherein said vectorfurther includes a promoter which is not a tissue-specific promoter. 16.The vector of claim 15 wherein said promoter is a Rous Sarcoma Viruspromoter.
 17. The vector of claim 14 wherein said vector furtherincludes at least one genomic element.
 18. The vector of claim 17wherein said genomic element is the full 3' untranslated region of theFactor IX DNA sequence.
 19. The vector of claim 17 wherein said genomicelement is the full 5' untranslated region of the Factor IX DNAsequence.
 20. The vector of claim 19 wherein said vector furtherincludes at least a portion of an intron of the Factor IX gene.
 21. Thevector of claim 20 wherein said at least a portion of an intron of theFactor IX gene is a centrally truncated first intron of the Factor IXgene, wherein said centrally truncated first intron includes the first991 base pairs of the Factor IX first intron and the last 448 base pairsof the Factor IX first intron.
 22. The vector of claim 21 wherein saidvector further includes the full 3' untranslated region of the Factor IXDNA sequence.
 23. The vector of claim 17 wherein said genomic element isat least a portion of an intron of the Factor IX gene.
 24. The vector ofclaim 23 wherein said at least a portion of an intron of the Factor IXgene is a centrally truncated first intron of the Factor IX gene,wherein said centrally truncated first intron includes the first 991base pairs of the Factor IX first intron and the last 448 base pairs ofthe Factor IX first intron.
 25. The vector of claim 17 wherein saidvector further includes the full seventh intron of the Factor IX gene.26. The vector of claim 14 wherein said Factor IX or a fragment thereofis human Factor IX or a fragment thereof having Factor IX clottingactivity.
 27. The vector of claim 1 wherein said vector comprisesadenoviral 5' ITR; an adenoviral 3' ITR; an adenoviral encapsidationsignal; at least one DNA sequence encoding a clotting factor; and apromoter controlling said at least one DNA sequence encoding a clottingfactor, wherein said vector is free of at least the majority ofadenoviral E1 and E3 DNA sequences, and is not free of all of the E2 andE4 DNA sequences, and DNA sequences encoding adenoviral proteinspromoted by the adenoviral major late promoter.
 28. The vector of claim27 wherein said vector also is free of at least a portion of at leastone DNA sequence selected from the group consisting of the E2 and E4 DNAsequences.
 29. The vector of claim 28 wherein said vector also is freeof at least the majority of the adenoviral E1 and E3 DNA sequences, andis free of a portion of the other of the E2 and E4 sequences.
 30. A celltransduced with the vector of claim
 1. 31. The cell of claim 30 whereinsaid DNA sequence encodes Factor VIII or a fragment thereof havingFactor VIII clotting activity.
 32. The cell of claim 31 wherein saidFactor VIII is human Factor VIII or a fragment thereof having FactorVIII clotting activity.
 33. The cell of claim 31 wherein said DNAsequence encodes B domain deleted Factor VIII.
 34. The cell of claim 33wherein said B domain is deleted Factor VIII is human B domain deletedFactor VIII.
 35. The cell of claim 33 wherein said vector furtherincludes a tissue-specific promoter.
 36. The cell of claim 35 whereinsaid tissue-specific promoter is the mouse albumin promoter.
 37. Thecell of claim 36 wherein said vector further includes the first exon ofthe apolipoprotein A-1 gene, the first intron of the apolipoprotein A-1gene, and the upstream portion of the second exon of the apolipoproteinA-1 gene to the start codon of the apolipoprotein A-1 gene.
 38. The cellof claim 33 wherein said vector further includes at least one genomicelement.
 39. The cell of claim 38 wherein said vector includes the ApoA1promoter.
 40. The cell of claim 39 wherein said vector further includesthe first exon of the apolipoprotein A-1 gene, the first intron of theapolipoprotein A-1 gene, and the upstream portion of the second exon ofthe apolipoprotein A-1 gene to the start codon of the apolipoprotein A-1gene.
 41. The cell of claim 30 wherein said DNA sequence encodes FactorIX or a fragment thereof having Factor IX clotting activity.
 42. Thecell of claim 41 wherein said Factor IX is human Factor IX or a fragmentthereof having Factor IX clotting activity.
 43. The cell of claim 41wherein said vector further includes a promoter which is not atissue-specific promoter.
 44. The cell of claim 43 wherein said promoteris a Rous Sarcoma Virus promoter.
 45. The cell of claim 43 wherein saidvector further includes at least one genomic element.
 46. The cell ofclaim 45 wherein said genomic element is the full 3' untranslated regionof the Factor IX DNA sequence.
 47. The cell of claim 45 wherein saidgenomic element is the full 5' untranslated region of the Factor IX DNAsequence.
 48. The cell of claim 47 wherein said vector further includesat least a portion of an intron of the Factor IX gene.
 49. The cell ofclaim 48 wherein said at least a portion of an intron of the Factor IXgene is a centrally truncated first intron of the Factor IX gene,wherein said centrally truncated first intron includes the first 991base pairs of the Factor IX first intron and the last 448 base pairs ofthe Factor IX first intron.
 50. The cell of claim 49 wherein said vectorfurther includes the full 3' untranslated region of the Factor IX DNAsequence.
 51. The cell of claim 45 wherein said vector further includesthe full seventh intron of the Factor IX gene.
 52. The cell of claim 45wherein said genomic element is at least a portion of an intron of theFactor IX gene.
 53. The cell of claim 52 wherein said at least a portionof an intron of the Factor IX gene is a centrally truncated first intronof the Factor IX gene, wherein said centrally truncated first intronincludes the first 991 base pairs of the Factor IX first intron and thelast 448 base pairs of the Factor IX first intron.
 54. A process forexpressing a clotting factor in a mammal, comprising:administering to amammal the vector of claim
 1. 55. The process of claim 54 wherein saidmammal is a human.
 56. The process of claim 55 wherein said vector isadministered intravenously.
 57. The process of claim 54 wherein saidclotting factor is Factor VIII or a fragment thereof having Factor VIIIclotting activity.
 58. The process of claim 57 wherein said Factor VIIIis human Factor VIII or a fragment thereof having Factor VIII clottingactivity.
 59. The process of claim 57 wherein said clotting factor is Bdomain deleted Factor VIII.
 60. The process of claim 59 wherein said Bdomain deleted Factor VIII is human B domain deleted Factor VIII. 61.The process of claim 59 wherein a sufficient amount of said vector isadministered to a mammal to provide at least 10 ng/ml of B domaindeleted Factor VIII in the blood of said mammal.
 62. The process ofclaim 54 wherein said clotting factor is Factor IX or a fragment thereofhaving Factor IX clotting activity.
 63. The process of claim 62 whereinsaid Factor IX is human Factor IX or a fragment thereof having Factor IXclotting activity.
 64. The process of claim 62 wherein a sufficientamount of said vector is administered to a mammal to provide at least250 ng/ml of Factor IX in the blood of said mammal.
 65. The method ofclaim 54 wherein said vector comprises an adenoviral 5' ITR; anadenoviral 3' ITR; an adenoviral encapsidation signal; at least one DNAsequence encoding a clotting factor; and a promoter controlling said atleast one DNA sequence encoding a clotting factor, wherein said vectoris free of at least the majority of adenoviral E1 and E3 DNA sequences,and is not free of all of the E2 and E4 DNA sequences, and DNA sequencesencoding adenoviral proteins promoted by the adenoviral major latepromoter.